Apparatus for applying thermal energy to a receptacle and detecting an emission signal from the receptacle

ABSTRACT

An apparatus for performing nucleic acid amplification reactions includes a thermally-conductive receptacle holder with front and back surfaces. The front surface includes a row of receptacle wells, and an outer surface of each well has a frustoconical shape, an inner surface of each well receives a receptacle, and each well has a through-hole extending between the inner and outer surfaces thereof. One or more thermal elements in contact with the back surface of the holder alter a temperature(s) of the holder. The apparatus includes multiple optical fibers, and each optical fiber provides optical communication between one of the wells and an excitation signal source and/or an emission signal detector. A first end of each optical fiber is disposed outside, within, or extends through the through-hole of a corresponding well, and a second end of each optical fiber is in optical communication with the excitation signal source and/or the emission signal detector.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application claiming the benefitunder 35 U.S.C. § 120 of the filing date of non-provisional patentapplication Ser. No. 16/434,910 filed Jun. 7, 2019, now U.S. Pat. No.10,961,572, which is a continuation application claiming the benefitunder 35 U.S.C. § 120 of the filing date of non-provisional patentapplication Ser. No. 13/956,022 filed Jul. 31, 2013, now U.S. Pat. No.10,494,668, which claims the benefit under 35 U.S.C. § 119(e) of thefiling date of provisional patent application Ser. Nos. 61/677,976,filed Jul. 31, 2012, and 61/783,952, filed Mar. 14, 2013, the respectivedisclosures of which are incorporated herein by reference.

BACKGROUND

The present disclosure relates generally to a system and apparatus forautomated heating and/or cooling of samples.

BACKGROUND INFORMATION

Automated molecular assay instrumentation offers numerous advantages,however most automated instruments suffer from a limited set of assaycapabilities. These limited capabilities complicate or inhibit parallelprocessing of multiple assays and, as a result, reduce sample throughputand flexibility in assay choices. This is particularly true for assaysrequiring incubation of some sort, such as the temperature cyclingnecessary for polymerase chain reaction (PCR) based assays. In PCRinstruments a thermocycler is included that is capable of cycling thetemperature of many samples, such as a batch of samples held within a96-well microtiter plate. This assay format requires the preparation ofthe entire batch of samples prior to subjecting them to an initialtemperature cycle. For example, the first sample that is fully preparedfor temperature cycling must wait until the last sample is preparedprior to temperature cycling. In the case of a 96-well plate, this waittime can be substantial, thus slowing the throughput of the instrument.In addition, since all samples are subject to the same temperatureprofile and cycling parameters, the types of assays that can be run inparallel are limited. Different assays often must be run in completelydifferent thermocycler units or await availability of the thermocyclerfrom a prior batch of samples. This too inhibits the ability to providerapid assay results.

The present disclosure addresses these and other needs in the art.

All documents referred to herein, or the indicated portions, are herebyincorporated by reference herein. No document, however, is admitted tobe prior art to the claimed subject matter.

SUMMARY

The present disclosure relates to a system and apparatus for alteringthe temperature of at least one receptacle holder that is adapted foruse in an automated instrument capable of performing biochemical assays.

In an aspect of the present disclosure, there is provided an apparatusthat includes one or more receptacle holders made of a heat-conductingmaterial. Each receptacle holder includes a plurality of receptaclewells, each receptacle well being configured to receive a receptacletherein; a plurality of through-holes, each through-hole extending froman inner surface of one of the receptacle wells to an outer surface ofthe receptacle holder; a plurality of optical fibers, each optical fiberhaving first and second ends, wherein the first end is in opticalcommunication with one of the receptacle wells, and the second end is inoptical communication with at least one of an excitation signal sourceand an emission signal detector, the first end of each optical fiberbeing disposed outside, within, or extending through, a correspondingthrough-hole in each receptacle well, and wherein the first end of eachoptical fiber is moveable or fixedly disposed within one of thethrough-holes relative to the surface of the receptacle well; and one ormore thermal elements positioned proximal to the receptacle holder foraltering a temperature or temperatures of the receptacle holder. Incertain embodiments, the apparatus includes a cover movable between anopened position and a closed position relative to the receptacle holder,wherein one or more receptacles disposed within one or more of thereceptacle wells are seated or secured into the receptacle well by thecover when the cover is moved into the closed position. In an exemplaryembodiment, the first end of each optical fiber moves within itscorresponding through-hole when (1) the cover is moved to the closedand/or opened position, or (2) a receptacle is present in the receptaclewell and the cover is moved to the closed and/or open position. In avariety of these embodiments, the apparatus does not include a cover.Also, in a variety of these embodiments, when these apparatuses arecomprised in a system, one or more of these apparatuses do/does notinclude a cover. One or more receptacle transport mechanism(s) are oftenincluded in such systems to transport receptacles to the receptacleswells, deposit the receptacles in the receptacles wells, optionallyensure each receptacle is securely seated in its respective receptaclewell by using, for example, physical contact, and removing eachreceptacle from its respective receptacle well. More than one receptacletransport mechanism may be utilized in such embodiments to effect one ormore of these steps.

In various embodiments, the thermal element is positioned proximal to aside surface of the receptacle holder and provides thermal energythrough the receptacle holder to each of the plurality of receptaclewells. The thermal energy may be uniform. In frequent embodiments, theapparatus further includes one or more support(s), each positionedproximal to a side surface of one or more of the receptacle holders, andthe thermal element is positioned between the support and the receptacleholder. One or more thermistors may be disposed in contact with thereceptacle holder. In a variety of embodiments, the one or morethermistors and/or associated wiring thereof are disposed within achannel formed in the receptacle holder. In certain embodiments, theapparatus further includes one or more closed-ended channels formed onopposing sides of a receptacle well of the receptacle holder, eachhaving one of the thermistors disposed therein.

In certain embodiments the apparatus further includes one or morecross-braces positioned to provide compressive force between thereceptacle holder and the support. In other embodiments, the apparatusfurther includes one or more bodies having a low thermal conductivity,each connected directly or indirectly with a linker, positioned toprovide compressive force between the receptacle holder and the support.In various embodiments, the support of the apparatus is a heat sink orthe support is provided in thermal communication with a heat sink. Theapparatus may include a first controller electrically connected to thethermal element to cycle the temperature of the thermal element and mayinclude one or more motors electrically connected to the firstcontroller disposed in moveable communication with the cover, ifpresent. In various embodiments, if present, the cover may include arigid element and one or more flexible extensions, wherein the flexibleextensions are attached to, and extend laterally away from, the rigidelement to apply a force when the cover is in the closed position to atleast a portion of one or more receptacles when present within thereceptacle wells.

In exemplary embodiments, the apparatus further includes a stripperplate in movable association with the receptacle holder for removing areceptacle from a receptacle transport mechanism that delivers areceptacle to the receptacle holder. The stripper plate may be moveablerelative to the receptacle well into unlocked and locked positions,wherein the unlocked position permits access of a receptacle to thereceptacle well, and wherein the locked position inhibits removal of thereceptacle from the receptacle well without inhibiting access to thereceptacle by the receptacle transport mechanism.

In another aspect, the disclosure provides a system that includes one ormore apparatus of the present disclosure. In various embodiments, eachof the one or more apparatus is in independent thermal communicationwith a single heat sink. The thermal element corresponding to eachreceptacle holder within the system may be independently controllable toonly alter the temperature of its corresponding receptacle holder. Infrequent embodiments, the system includes one or more controllerselectrically connected to the thermal elements, and to one or moremotors disposed in moveable communication with a cover corresponding toeach receptacle holder. In various embodiments, the system does notinclude a cover. The system may include at least ten receptacle wellsand at least ten corresponding optical fibers, wherein the second endsof all of the optical fibers are in optical communication with one ormore excitation signal sources and/or one or more emission signaldetectors. In frequent embodiments, the system is disposed in a singlehousing.

In another aspect, the disclosure provides a method of conducting anautomated, random-access incubation process. In one exemplaryembodiment, the method includes the automated steps of transferring afirst set of receptacles to a first receptacle holder and subjecting thecontents of the first set of receptacles to a first incubation process,and during the first incubation process, transferring a second set ofreceptacles to a second receptacle holder and subjecting the contents ofthe second set of receptacles to a second incubation process. In anotherexemplary embodiment, he first and second receptacle holders arecomponents of the apparatus as disclosed herein. Each of the first andsecond set of receptacles may be sealed to prevent contamination and/orevaporation. In various exemplary embodiments, the first and secondreceptacle holders are each in thermal communication with a singletemperature cycling apparatus, as described herein.

In frequent embodiments, the method further includes, during the secondincubation process, beginning a third of three or more independentprocesses comprising: transferring a third or higher set of receptaclesto a third or higher receptacle holder and subjecting the contents ofthe third or higher set of receptacles to a third or higher incubationprocess, wherein the transfer of each successive set of receptacles isbegun prior to completion of the incubation process for each immediatelypreceding set of receptacles. The transfer of the first and second setof receptacles may be effected by a receptacle transport mechanism. Asdescribed herein, each set of receptacles may be removed from itsrespective receptacle holder prior to completion of the next successiveincubation process.

In another aspect, the disclosure provides a method or establishingoptical communication between a receptacle and an excitation signalsource and/or an emission signal detector within a housing of anapparatus. The method includes the automated steps of providing areceptacle to a well of a receptacle holder comprised of aheat-conducting material, applying a first force to the receptacle,thereby seating the receptacle within the well, and either of (1) whilethe force is being applied to the receptacle, effecting movement of anend of an optical fiber toward and into contact with the seatedreceptacle, or (2) while the force is being applied to the receptacle,the receptacle applies a second force to an end of an optical fiberdisposed within the well such that the end of the optical fiber moveswithin the well in a direction opposite from the direction of theapplied second force.

In various embodiments, the receptacle is in contact with the opticalfiber during or after step (a), and while the force is being applied tothe receptacle, the receptacle contacts and applies force to the end ofthe optical fiber disposed within the well such that the end of theoptical fiber moves within the well in a direction opposite from thedirection of the applied force. The end of or an area proximal to theend of the optical fiber may be connected, directly or indirectly, witha resilient element to the receptacle holder such that the resilientelement compresses when force is applied to the receptacle.

In yet another exemplary aspect, the disclosure provides an apparatusthat includes a housing, a plurality of receptacle holders containedwithin the housing, each comprised of a heat-conducting material,wherein each receptacle holder comprises a plurality of receptaclewells, one or more thermal elements positioned proximal to eachreceptacle holder for altering a temperature or temperatures of theplurality of receptacle wells. In various embodiments, the apparatusfurther includes a plurality of covers, each disposed in moveableassociation with a receptacle holder, wherein a first controllercontrols movement of each cover between an opened and a closed position.When at least one receptacle is present in the plurality of receptaclewells it is secured within the receptacle wells by the cover when in theclosed position. When the cover is in the opened position, a receptacletransport mechanism can access the plurality of receptacle wells tointroduce or remove a receptacle. In addition, each cover can movebetween the opened and closed positions together with, or independentlyof, one or more other covers. In other various embodiments, theapparatus does not include a cover.

In various embodiments, the apparatus further includes an optical fiberassociated with each receptacle well such that optical communication isestablished between an interior of each receptacle well and anexcitation signal source and/or an emission signal detector. Theapparatus may further include one or more receptacles in the pluralityof receptacle wells, and optical communication is established betweeneach receptacle and the excitation signal source and/or the emissionsignal detector by the optical fibers. In frequent embodiments, theapparatus further includes a stripper plate in moveable association witheach receptacle holder for removing a receptacle from the receptacletransport mechanism.

In various embodiments, the support of the apparatus is a heat sink orthe support is provided in thermal communication with a heat sink. Theapparatus may include a first controller electrically connected to thethermal element to cycle the temperature of the thermal element and mayinclude one or more motors electrically connected to the firstcontroller disposed in moveable communication with the cover, ifpresent. In various embodiments, if present, the cover may include arigid element and one or more flexible extensions, wherein the flexibleextensions are attached to, and extend laterally away from, the rigidelement to apply a force when the cover is in the closed position to atleast a portion of one or more receptacles when present within thereceptacle wells.

In yet another exemplary aspect, the disclosure provides an apparatusthat includes a housing, a plurality of receptacle holders containedwithin the housing, each comprised of a heat-conducting material,wherein each receptacle holder comprising a plurality of receptaclewells, and one or more thermal elements electrically connected to afirst controller, and positioned proximal to each receptacle holder foraltering a temperature or temperatures of the plurality of receptaclewells. In various embodiments, the apparatus may further include aplurality of covers, each being movable between a first non-engagementposition and a second engagement position with respect to a receptacleholder, wherein each cover comprises a series of flexible extensions,wherein each individual flexible extension is associated with a singlereceptacle well within the receptacle holder, and wherein the movementof each cover between the first non-engagement position and secondengagement position is controlled by a second controller. When at leastone receptacle is present in the plurality of receptacle wells it issecured within the receptacle well by the cover when in the secondposition. When the cover is in the first position, a receptacletransport mechanism can access the plurality of receptacle wells tointroduce or remove a receptacle. In addition, each cover can movebetween the first and second positions together with, or independentlyof, one or more other covers. The first controller and the secondcontroller may be the same unit. In other various embodiments, theapparatus does not include a cover or a plurality of covers.

In various embodiments, the support of the apparatus is a heat sink orthe support is provided in thermal communication with a heat sink. Theapparatus may include a first controller electrically connected to thethermal element to cycle the temperature of the thermal element and mayinclude one or more motors electrically connected to the firstcontroller disposed in moveable communication with the cover, ifpresent. In various embodiments, if present, the cover may include arigid element and one or more flexible extensions, wherein the flexibleextensions are attached to, and extend laterally away from, the rigidelement to apply a force when the cover is in the closed position to atleast a portion of one or more receptacles when present within thereceptacle wells.

In yet another exemplary aspect, the disclosure provides an apparatusthat includes one or more receptacle holders made of a heat-conductingmaterial. Each receptacle holder includes a plurality of receptaclewells, each receptacle well being configured to receive a receptacletherein; a plurality of through-holes, each through-hole extending froman inner surface of one of the receptacle wells to an outer surface ofthe receptacle holder; and a plurality of optical fibers, each opticalfiber having first and second ends, wherein the first end is in opticalcommunication with one of the receptacle wells, and the second end is inoptical communication with at least one of an excitation signal sourceand/or an emission signal detector, the first end of each optical fiberbeing disposed outside, within, or extending through, a correspondingthrough-hole in each receptacle well, one or more thermal elementspositioned proximal to the receptacle holder for altering a temperatureor temperatures of the receptacle holder. In various embodiments, theapparatus does not have a cover. In various embodiments, the apparatusfurther includes a primary cover fixedly positioned over the receptacleholder and having one or more securing arms in alignment with anddisposed in a surrounding arrangement with each receptacle well of thereceptacle holder, wherein one or more receptacles disposed within oneor more of the receptacle wells are seated or secured into thereceptacle well by the securing arms; and a secondary cover fixedlypositioned over the primary cover and having one or more releasing armsin alignment with and in sliding contact with the securing arms of theprimary cover. In an exemplary embodiment, application of a force ontothe releasing arms urges the securing arms to flex in a radial outwarddirection relative to an axial center of the receptacle well, therebyreleasing the receptacle disposed within the receptacle well. One, two,three, four, or more securing arms are contemplated. In other variousembodiments, the apparatus does not include a primary or secondarycover. In a variety of embodiments, the one or more thermistors and/orassociated wiring thereof are disposed within a channel formed in thereceptacle holder. In certain embodiments, the apparatus furtherincludes one or more closed-ended channels formed on opposing sides of areceptacle well of the receptacle holder, each having one of thethermistors disposed therein.

In yet another exemplary aspect, the disclosure provides an apparatusthat includes one or more receptacle holders made of a heat-conductingmaterial. Each receptacle holder includes a plurality of receptaclewells, each receptacle well being configured to receive a receptacletherein; a plurality of through-holes, each through-hole extending froman inner surface of one of the receptacle wells to an outer surface ofthe receptacle holder; a plurality of optical fibers, each optical fiberhaving first and second ends, wherein the first end is in opticalcommunication with one of the receptacle wells, and the second end is inoptical communication with at least one of an excitation signal sourceand an emission signal detector, the first end of each optical fiberbeing disposed outside, within, or extending through, a correspondingthrough-hole in each receptacle well, and wherein the first end of eachoptical fiber is moveable within one of the through-holes relative tothe surface of the receptacle well; and one or more thermal elementspositioned proximal to the receptacle holder for altering a temperatureor temperatures of the receptacle holder. In various embodiments, theapparatus may further include a cover movable between an opened positionand a closed position relative to the receptacle holder. When present,one or more receptacles disposed within one or more of the receptaclewells are securely seated to maximize contact with the inner surfaces ofthe receptacle wells without the need for contact with the cover. Incertain embodiments, the apparatus may not include a cover. In a varietyof embodiments, the one or more thermistors and/or associated wiringthereof are disposed within a channel formed in the receptacle holder.In certain embodiments, the apparatus further includes one or moreclosed-ended channels formed on opposing sides of a receptacle well ofthe receptacle holder, each having one of the thermistors disposedtherein.

In other embodiments, the present disclosure provides methods ofintroducing and removing a receptacle from a receptacle holder utilizinga fluid transfer apparatus, wherein the fluid transfer apparatus isconfigured to fixedly introduce the receptacle to the receptacle holderand to release the receptacle from a securing mechanism disposed withinthe receptacle holder that fixedly holds the receptacle within thereceptacle holder.

In yet another exemplary aspect, the disclosure provides a system thatincludes one or more apparatus of the present disclosure. In variousembodiments, the system also includes a receptacle transport mechanism,which may be a modified pipettor. The receptacle transport mechanismincludes a body having a plunger slidingly disposed therein, and one ormore limbs hingedly attached to the body and positioned in slidingcommunication with a knob fixedly attached to the plunger. When theplunger is in a first position, a lower portion of the one or more limbsare proximal to the body, and when the plunger is in a second position,the lower portion of the one or more limbs are extended in a radialoutward direction relative to the body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial diagram showing an apparatus of the presentdisclosure.

FIG. 2 is a pictorial diagram showing an apparatus of the presentdisclosure mounted in a housing.

FIGS. 3A-3C are pictorial diagrams showing a receptacle holder of thepresent disclosure.

FIG. 4 is a pictorial diagram showing the top view of an apparatus ofthe present disclosure mounted in a housing.

FIGS. 5A-5C are pictorial diagrams showing a receptacle holder mountedin sliding engagement with a support. The support is mounted in thermalcommunication with a heat sink (FIG. 5A). A cross-brace may be mountedto the support to exert a force onto a front surface the receptacleholder (FIG. 5B). A detailed view of the support is shown in FIG. 5C.

FIGS. 5D and 5E are pictorial diagrams showing an exemplary receptacleholder. The receptacle holder may include a channel through which thewiring and/or electrical connections for one or more thermistors may bedisposed (FIG. 5D). The receptacle holder may also include one or morebulges formed corresponding to closed through-holes disposed within thechannel for containing the one or more thermistors (FIG. 5E).

FIGS. 6A and 6B are pictorial diagrams showing a receptacle holdermounted in sliding engagement with a support.

FIGS. 7A and 7B are pictorial diagrams showing multiple rows ofreceptacle holders disposed in an apparatus of the present disclosure(FIG. 7A), and that cartridge heaters may be disposed within the heatsink of the apparatus (FIG. 7B).

FIGS. 8A-8E are pictorial diagrams showing exemplary covers and stripperplates disposed within the apparatus of the present disclosure.

FIGS. 9A-9C are pictorial diagrams showing movement of the opticalfibers of the apparatus of the present disclosure and the forcesassociated therewith prior to and after seating receptacles within thereceptacle wells of a receptacle holder.

FIG. 10 is a pictorial diagram showing an apparatus in opticalcommunication with an excitation signal source and/or an emission signaldetector within a housing of an instrument for performing a biochemicalassay.

FIGS. 11A and 11B are flow charts showing exemplary steps involved in amethod for establishing optical communication between a receptacle andan excitation signal source and/or an emission signal detector within ahousing of the apparatus while allowing maximal contact between thesurface of the receptacle well and the receptacle.

FIGS. 12A-12D are pictorial diagrams showing exemplary steps involved inloading receptacles into the receptacle wells of a receptacle holder ofan apparatus of the present disclosure.

FIG. 13 is a flow chart showing exemplary steps involved in a method ofa method of conducting automated, random-access temperature cyclingprocesses.

FIGS. 14A-14D are pictorial diagrams showing a second exemplaryembodiment of an apparatus of the present disclosure.

FIGS. 15A-15C are pictorial diagrams showing a modified pipettor for useas a receptacle transport mechanism within a system of the presentdisclosure.

FIG. 16 is a pictorial diagram showing a perspective view of analternative exemplary embodiment of the cover mechanism of theapparatus.

DETAILED DESCRIPTION

The present disclosure relates to a system, apparatus, and method forincubating at least one receptacle holder that is adapted for use in anautomated instrument capable of performing nucleic acid-basedamplification assays. Also provided are methods for conductingautomated, random-access temperature cycling processes using the same.

Before the present systems, methods, and apparatuses are described, itis to be understood that this disclosure is not limited to particularmethods and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting, since the scope of the presentdisclosure will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

The term “comprising,” which is used interchangeably with “including,”“containing,” “having,” or “characterized by,” is inclusive oropen-ended language and does not exclude additional, unrecited elementsor method steps. The phrase “consisting of” excludes any element, step,or ingredient not specified in the claim. The phrase “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristics of the disclosed subject matter. The present disclosurecontemplates exemplary embodiments of an apparatus and methods of usethereof corresponding to the scope of each of these phrases. Thus, anapparatus or method comprising recited elements or steps contemplatesparticular embodiments in which the apparatus or method consistsessentially of or consists of those elements or steps.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing disclosed herein, the preferred methods andmaterials are now described.

As used herein, a “reaction mixture” refers to a volume of fluidcomprising one or more of a buffer for a nucleic acid amplificationreaction, one or more nucleotides, an enzyme, and a sample containing orsuspected of containing a nucleic acid.

As used herein, a “sample” or a “test sample” refers to any substancesuspected of containing a target organism or biological molecule, suchas nucleic acid. The substance may be, for example, an unprocessedclinical specimen, a buffered medium containing the specimen, a mediumcontaining the specimen and lytic agents for releasing nucleic acidbelonging to the target organism, or a medium containing nucleic acidderived from a target organism which has been isolated and/or purifiedin a reaction receptacle or on a reaction material or device. In someinstances, a sample or test sample may comprise a product of abiological specimen, such as an amplified nucleic acid to be detected.

As used herein, “analyte” refers to a substance, such as a nucleic acidor protein, that is detected or measured in an analytical procedure. Theanalyte may be contained in a sample undergoing testing.

As used herein, “polynucleotide” refers to either RNA, DNA, or achimeric molecule containing both RNA and DNA.

As used herein, “nucleic acid” refers to a multimeric compoundcomprising nucleosides or nucleoside analogs which have nitrogenousheterocyclic bases, or base analogs, which are linked by phosphodiesterbonds or other linkages to form a polynucleotide. Nucleic acids includeRNA, DNA, or chimeric DNA-RNA polymers, and analogs thereof. A nucleicacid “backbone” may be made up of a variety of linkages, including oneor more of sugar-phosphodiester linkages, peptide-nucleic acid (PNA)bonds (PCT No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties ofthe nucleic acid may be either ribose or deoxyribose, or similarcompounds having known substitutions, such as 2′ methoxy substitutionsand 2′ halide substitutions (e.g., 2′-F). Nitrogenous bases may beconventional bases (A, G, C, T, U), analogs thereof (e.g., inosine),derivatives of purine or pyrimidine bases, such as N⁴-methyldeoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or replacement substituent at the 2, 6and/or 8 position, such as 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine (U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” positions in whichthe backbone does not include a nitrogenous base for one or moreresidues (see U.S. Pat. No. 5,585,481). Nucleic acids also include“locked nucleic acids” (LNA), an analog containing one or more LNAnucleotide monomers with a bicyclic furanose unit locked in an RNAmimicking sugar conformation (Vester et al., 2004, Biochemistry43(42):13233-41). A nucleic acid may comprise only conventional sugars,bases, and linkages as found in RNA and DNA, or may include conventionalcomponents and substitutions (e.g., conventional bases linked by a 2′methoxy backbone, or a nucleic acid including a mixture of conventionalbases and one or more base analogs). Methods for synthesizing nucleicacids in vitro are well known in the art.

As used herein “oligonucleotide” or “oligomer” refers to a polymer madeup of two or more nucleoside subunits or nucleobase subunits coupledtogether. Oligonucleotides preferably have a length in the range of from10-100 nucleotides, more preferably 10-80 nucleotides, and still morepreferably from 15-60 nucleotides. The oligonucleotide may be DNA and/orRNA and analogs thereof. The sugar groups of the nucleoside subunits maybe ribose, deoxyribose and analogs thereof, including, for example,ribonucleosides having a 2′-O-methylsubstitution to the ribofuranosylmoiety. Oligonucleotides including nucleoside subunits having 2′substitutions and which are useful as detection probes, capture oligosand/or amplification oligonucleotides are disclosed in U.S. Pat. No.6,130,038. The nucleoside subunits may be joined by linkages such asphosphodiester linkages, modified linkages, or by non-nucleotidemoieties, which do not prevent hybridization of the oligonucleotide toits complementary target nucleic acid sequence. Modified linkagesinclude those linkages in which a standard phosphodiester linkage isreplaced with a different linkage, such as a phosphorothioate linkage ora methylphosphonate linkage. The nucleobase subunits may be joined, forexample, by replacing the natural deoxyribose phosphate backbone of DNAwith a pseudo-peptide backbone, such as a 2-aminoethylglycine backbonewhich couples the nucleobase subunits by means of a carboxymethyl linkerto the central secondary amine. (DNA analogs having a pseudo-peptidebackbone are commonly referred to as “peptide nucleic acids” or “PNA,”and are disclosed in U.S. Pat. No. 5,539,082). Other non-limitingexamples of oligonucleotides or oligomers contemplated by the presentdisclosure include nucleic acid analogs containing bicyclic andtricyclic nucleoside and nucleotide analogs referred to as “LockedNucleic Acids,” “Locked Nucleoside Analogues” or “LNA.” (see, e.g., U.S.Pat. Nos. 6,083,482; 6,268,490; and 6,670,461). Any nucleic acid analogis contemplated by the present disclosure, provided that the modifiedoligonucleotide can hybridize to a target nucleic acid under eitherstringent hybridization conditions or amplification reaction conditions.

As used herein, the term “biochemical assay” refers to a scientificinvestigative procedure for qualitatively assessing or quantitativelymeasuring the presence or amount or the functional activity of a targetentity, such as, but not limited to, a biochemical substance, a cell,organic sample, or target nucleic acid sequence. Included in the term“biochemical assay” are nucleic acid amplification and heat denaturation(i.e., melting). Nucleic acid melting typically involves precise warmingof a double stranded nucleic acid molecule to a temperature at which thetwo strands separate or “melt” apart. The melting process typicallyoccurs at a temperature of about 50° C. to about 95° C.

As used herein, a “target nucleic acid sequence” or “target sequence”refers to a strand of nucleic acid molecules that is be assayed. Thus,when used in the context of an amplification assay, a target sequencerefers to a sequence of nucleotides or a portion of a nucleic acidmolecule that is intended to be copied.

As used herein, “amplification” or “amplifying” refers to an in vitroprocedure for obtaining multiple copies of a target nucleic acidsequence, its complement or fragments thereof. For example, an in vitroamplification reaction is an enzyme-catalyzed reaction that results inthe synthesis of multiple copies of a target nucleic acid sequence, itscomplement or fragments thereof. Examples of amplification methods thatcan be used for preparing in vitro amplification reactions are givenbelow. Preferred in vitro amplification reactions synthesize ampliconsin an exponential fashion, meaning that one amplicon serves as thetemplate for production of new amplicons. As used herein, the term“amplicon” or “amplification product” refers to a nucleic acid moleculegenerated in a nucleic acid amplification reaction. An amplicon oramplification product contains a target nucleic acid sequence that maybe of the same or opposite sense as the target nucleic acid.

Target nucleic acid amplification involves the use of amplificationoligonucleotides (e.g., primer sequences) and enzymes (e.g.,polymerases) to synthesize nucleic acid amplification products (copies)containing a sequence that is either complementary or homologous to thetemplate nucleic acid sequence being amplified. As used herein, an“amplification oligonucleotide” refers to a strand of nucleic acid thatserves as a starting point for the production of amplification products.The amplification products may be either extension products ortranscripts generated in a transcription-based amplification procedure.The amplification oligonucleotides may be provided to a reaction mixturefree in solution or one or more of the amplification oligonucleotidesmay be immobilized on a solid support, including the inner surface of achamber or chambers within a receptacle. See, e.g., U.S. Pat. Nos.5,641,658 and 7,582,470. Examples of nucleic acid amplificationprocedures practiced in the art include, but are not limited to, thepolymerase chain reaction (PCR), strand displacement amplification(SDA), helicase dependent amplification (HDA), loop-mediated isothermalamplification (LAMP), and a variety of transcription-based amplificationprocedures, including transcription-mediated amplification (TMA),nucleic acid sequence based amplification (NASBA), and self-sustainedsequence replication (3SR). See, e.g., Mullis, “Process for Amplifying,Detecting, and/or Cloning Nucleic Acid Sequences,” U.S. Pat. No.4,683,195; Walker, “Strand Displacement Amplification,” U.S. Pat. No.5,455,166; Kong et al., “Helicase Dependent Amplification of NucleicAcids,” U.S. Pat. No. 7,282,328, Notomi et al., “Process forSynthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278; Kacian et al.,“Nucleic Acid Sequence Amplification Methods,” U.S. Pat. No. 5,399,491;Becker et al., “Single-Primer Nucleic Acid Amplification Methods,” U.S.Pat. No. 7,374,885; Malek et al., “Enhanced Nucleic Acid AmplificationProcess,” U.S. Pat. No. 5,130,238; and Lizardi et al. (1988)BioTechnology 6:1197. With some procedures, the formation of detectableamplification products depends on an initial antibody/antigeninteraction. See, e.g., Cashman, “Blocked-Polymerase PolynucleotideImmunoassay Method and Kit,” U.S. Pat. No. 5,849,478. Nucleic acidamplification is especially beneficial when the amount of analyte (e.g.,targeted nucleic acid, antigen or antibody) present in a sample is verylow. By amplifying a target sequence associated with the analyte anddetecting the synthesized amplification product, the sensitivity of anassay can be vastly improved, since less analyte is needed at thebeginning of the assay to ensure detection of the analyte.

As used herein, the term “incubate,” “incubation,” “incubation process”and all variants thereof, refer collectively to altering the temperatureof an object in a controlled manner such that conditions are sufficientfor conducting the desired biochemical assay. Thus, it is envisionedthat the terms encompass heating a receptacle to a desired temperatureand maintaining such temperature for a fixed time interval. Alsoincluded in the terms is the act of subjecting a receptacle to one ormore heating and cooling cycles (i.e., “temperature cycling”). Whiletemperature cycling typically occurs at relatively high rates of changein temperature, the term is not limited thereto, and may encompass anyrate of change in temperature.

As used herein, a “detectable label,” or simply “label,” refers to achemical moiety that can be detected or can lead to a detectableresponse. Detectable labels in accordance with the present disclosurecan be linked to probes, such as hybridization probes, either directlyor indirectly. Examples of detectable labels include, but are notlimited, to, radioisotopes, enzymes, haptens, chromophores such as dyesor particles that impart a detectable color (e.g., latex beads or metalparticles), luminescent compounds (e.g., bioluminescent, phosphorescentor chemiluminescent moieties) and fluorescent compounds.

Receptacle Holder

The conditions of a nucleic acid amplification reaction may besubstantially isothermal or they may require periodic temperaturechanges, as with PCR thermal cycling. The apparatus described herein maybe used to heat and maintain a nucleic acid containing sample to aconstant or ambient temperature or it may be used to fluctuate thetemperature thereof. Target nucleic acid amplification reactions may beeither “real-time” or “end-point” assays. Accordingly, in an exemplaryaspect, there is provided an apparatus to perform the heating (i.e.,isothermal or temperature cycling) necessary for a nucleic acidamplification assay. As shown in FIG. 1 , the apparatus 100 includes oneor more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or any wholenumber between 1 and 20, or more) receptacle holders 110 (see also FIG.3 ). In an exemplary embodiment, the apparatus 100 includes two or morereceptacle holders 110. Such an apparatus may include a housing 50 (seeFIGS. 2 and 4 ) within which the one or more receptacle holders 110 arelocated. The housing 50 may be made from any suitable structuralmaterial such as, for example, plastic or metal.

When multiple receptacle holders 110 are provided in an apparatusdescribed herein, each receptacle holder 110 disposed within theapparatus may be disposed in alignment with one another to facilitatethe automated processing steps involved in nucleic acid amplificationassays. It should be understood that any alignment may be used inaccordance with the size and shape of the apparatus. In an exemplaryembodiment, the receptacle holders are disposed within the apparatus inone or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) rows of tworeceptacle holders per row, as shown in FIG. 4 . Thus, two receptacleholders may be disposed in a row either in thermal connection with, orthermally separated from, one another. In an exemplary embodiment, theapparatus 100 includes six rows of two receptacle holders per row.

As shown in FIGS. 3A-3C, the receptacle holder 110 includes a plurality(i.e., two or more) of receptacle wells 120 that are configured toreceive a receptacle 130 optionally containing a sample or reactionmixture 140. For purposes of explanation, the surface of the receptacleholder into which the receptacles 130 are inserted will be referred toas the “top surface” 150 thereof. Likewise, the surface of thereceptacle holder opposite to the surface into which the receptacles 130are inserted will be referred to as the “bottom surface” 160. In anexemplary embodiment, each receptacle holder 110 includes five or more(i.e., 5, 6, 7, 8, 9, 10, or any whole integer between 1 and 10, ormore) receptacle wells 120. In another exemplary embodiment, eachreceptacle holder 110 includes one to ten receptacle wells. In anotherexemplary embodiment, each receptacle holder includes three to sixreceptacle wells. In yet another exemplary embodiment, each receptacleholder includes five receptacle wells. Each of the plurality ofreceptacle wells within a respective receptacle holder may be disposedin alignment with one another. In an exemplary embodiment, thereceptacle wells 120 are disposed in a row extending along the length ofthe top surface 150 of the receptacle holder 110.

Exemplary materials from which a receptacle holder may be made include,but are not limited to, aluminum, titanium, copper, steel, magnesium,metal composites, metal alloys, ceramics, plastics, plastic composites,or any suitable thermally-conductive material.

As used herein, a receptacle well of the receptacle holder that is“configured to receive” a particular size or shape of receptacle refersto a receptacle well whose dimensions are substantially similar to thesize and shape of a receptacle 130 (i.e., a sample tube) such that thereceptacle 130 fits snugly within the receptacle well 120, therebymaximizing contact between the surface of the receptacle well 120 andthe receptacle 130. In certain embodiments, this maximal contact refersto physical contact of the receptacle well 120 with at least a portionof the receptacle 130. In various embodiments, receptacles 130 inaccordance with the present disclosure are individual reaction vesselsmade from suitable rigid or flexible materials, and shaped anddimensioned to fit within the receptacle wells of the apparatusdescribed herein. In other embodiments, two or more (i.e., 2, 3, 4, 5,or more) receptacles may be manufactured as a single unit configured tofit within a receptacle holder. Each receptacle 130 may be closed orsealed to prevent contamination of and/or evaporation of the contentstherein, and/or to facilitate handling or transport of each receptacle.Such seals may be permanent or semi-permanent and may be fluid-tight. Incertain embodiments the seal comprises a cap or lid 135.

Within each receptacle well 120 is at least one through-hole 170, whichextends from an inner surface 180 of the receptacle well to an outersurface of the receptacle holder. In an exemplary embodiment, thethrough-hole 170 of a particular receptacle well 120 is extends from thebottom-center of inner surface 180 of the receptacle well 120 andextends to the surface of the receptacle holder 110 that is opposite tothe surface of the receptacle holder within which the receptacles 130are inserted (i.e., in this embodiment, the through-hole extends fromthe bottom of the receptacle well 120 to the bottom surface 160 of thereceptacle holder 110). In certain embodiments the diameter of thethrough-hole 170 is the same as that of the bottom 190 of the innersurface 180 of receptacle well 120. In other embodiments, thethrough-hole 170 comprises a hole or opening having dimensions smallerthan the bottom 190 of the inner surface 180 of receptacle well 120. Inother embodiments, the through-hole 170 comprises a hole or openinghaving dimensions the same size as, or larger than, the bottom 190 ofthe inner surface 180 of receptacle well 120. The exact dimensions ofthe through-hole 170 may vary, provided that the presence of thethrough-hole 170 does not detrimentally affect the ability of thereceptacle holder 110 to efficiently transfer heat to and from areceptacle 130 held within the receptacle well 120.

Thermal Element

As shown in FIGS. 5A and 5B, positioned proximal to the receptacleholder is one or more thermal elements 200 for altering a temperature ortemperatures of the receptacle holder 110. As used herein, the term“thermal element” may include any known heating element for heating andcooling applications. In one embodiment, the thermal element is aresistive heating element, such as a thin metal film that is applied tothe receptacle holder 110 by using well-known methods such as sputteringor controlled vapor deposition. The heating element also can be providedas a molded or machined insert (e.g., such as a cartridge) forincorporation into the receptacle holder 110.

In an exemplary embodiment, the thermal element 200 is a thermoelectricdevice, such as a “Peltier device,” which is generally constructed fromelectron-doped n-p semiconductor pairs that act as miniature heat pumps.When current is applied to the semiconductor pairs, a temperaturedifference is established whereas one side becomes hot and the othercold. If the current direction is reversed, the hot and cold faces willbe reversed. Usually an electrically nonconductive material layer, suchas aluminum nitride or polyimide, is disposed over the substrate facesof the thermoelectric modules so as to allow for proper isolation of thesemiconductor element arrays.

As used herein, “altered temperature or temperatures” of the receptacleholder refers to the increase or decrease of the temperature of thereceptacle holder 110. Often, the increase or decrease of thetemperature is determined relative to the ambient temperature. Includedin the term is the ability to individually adjust the temperature of oneor more receptacle wells 120, while separately adjusting the temperatureof other receptacle wells within the same receptacle holder. Thus, theterm may refer to uniformly raising/lowering the temperature of allreceptacle wells 120 within a receptacle holder 110 or may refer toaltering a subset of the receptacle wells 120 within a single receptacleholder 110. As used herein, “ambient temperature” refers to thetemperature of a surrounding environment, which may include a fluid(e.g., air or liquid) or solid structure.

The thermal element 200 may be electrically connected to a controllablepower source 210 for applying a current across the element to alter thetemperature thereof. Control of the power source 210 can be carried outby an appropriately programmed controller 220 (such as a computer) whichreceives signals from one or more thermal sensors 610 (see FIG. 6A) inthermal communication with the receptacle holder 110, as discussedbelow, and/or signals from another processor 750 that controls theautomated process steps involved with temperature cycling processes.

The thermal element 200 may be held in contact with a side surface 115(see FIG. 3A) of the receptacle holder 110 by one or more supports 240(see FIG. 5C), which may be positioned in sliding engagement with thereceptacle holder 110. As used herein, being positioned “in slidingengagement” refers to a non-fixed contact between adjacent surfaces ofdifferent parts of the apparatus described herein. Thus, when theapparatus 100 includes two or more receptacle holders 110, each of thetwo or more receptacle holders are configured in sliding engagement witha support 240. As used herein, the term “support” refers to a rigidstructure, which can be thermally-conductive. Exemplary materials fromwhich a support may be made include, but are not limited to, aluminum,titanium, copper, steel, magnesium, metal composites, metal alloys,ceramics, plastics, plastic composites, or any suitable rigidthermally-conductive material. Supports may also comprise a structureformed of, or from, a combination of materials, for example, plastic,metal (including alloys and composites), ceramic, or a combination ofdifferent types of one or more of these materials.

As is known in the art, thermal elements may require a specific force toachieve adequate thermal contact with a component that is to be heated.For example, certain Peltier devices require a mounting force ofapproximately 150-300 psi to effectively transfer thermal energy to adevice. With reference to FIG. 5B, the apparatus may include one or morecross-braces 248 mounted to a support 240, and which exert a force F1onto a front surface 117 (see FIG. 3A) of a receptacle holder 110. ForceF1 is sufficient to effect thermal transfer of energy from thermalelement 200 to receptacle holder 200. In certain embodiments, theapparatus includes one cross-brace 248 for each receptacle holder 110.In other embodiments, the apparatus includes one cross-brace 248 per rowof receptacle holders 110. In such embodiments, the cross-bracegenerally incorporates a portion or layer having low thermalconductivity as the portion that directly contacts the receptacle holder110. As discussed below, in other embodiments, a body 300 (see FIGS. 6A,6B, 7A) having low thermal conductivity is used to exert the forcerequired for thermal transfer of energy to the receptacle holder 110.

Support

As shown in FIG. 5C, the support 240 may be formed in a shape suitablefor use in the apparatus described herein. In an exemplary embodiment,the support is a solid member having a base portion 245 and an uprightportion 247. In certain embodiments, the base portion 245 and uprightportion 247 comprise a single contiguous material. The upright portion247 may intersect the base portion at a right angle or may intersect thebase portion 245 at an angle greater or less than 90°. Disposed withinthe base portion is a plurality of through-holes 242, which arepreferably in alignment with the through-holes 170 of the bottom surface160 of the receptacle holder 110 that will be positioned in slidingengagement therewith. Each of the through-holes 242 of the base portion245 of the support 240 form a channel through which optical fibers 400(see FIGS. 5A, 7A) and/or associated components such as a fixed ormoveable ferrule 450, for example, may pass, thereby providing opticalcommunication between each receptacle well 120 and an excitation signalsource and/or an emission signal detector, as discussed below.

The upright portion 247 of the support 240 includes a first side 243 andsecond side 244. The first side 243 is configured to be positionedproximal to a side surface 115 of the receptacle holder 110, with thethermal element 200 being positioned between the first side 243 of thesupport 240 and the receptacle holder 110. The second side 244 of theupright portion 247 of the support 240 provides a solid surface to whichat least one body 300 having low thermal conductivity may be disposed(see FIGS. 6A, 6B, and 7A).

Body & Linker

As shown in FIGS. 6A, 6B, and 7A, when the apparatus 100 includesmultiple supports 240 that are oriented in rows, as discussed above, theapparatus may include one or more bodies 300 having low thermalconductivity, each associated with a respective receptacle well 120 of areceptacle holder 110. In certain embodiments, the apparatus willinclude a linker for exerting force F1 through the body 300 onto thereceptacle holder 110.

As used herein, the term “linker” refers to any device that can exert aforce in the direction that extends away from the surface upon which itis mounted. Exemplary linkers useful in the apparatus include, but arenot limited to, springs, spacers, linear expanders, materials formed ofelastic or rubbery material, piezoelectric devices, levers, screws, etc.As such, in an exemplary embodiment, a first end 312 of a linker 310(e.g., spring) is mounted to the second side 243 of the upright portion247 of a support 240, while a second end 314 contacts and exerts a forceF1 onto a body 300 having lower thermal conductivity (e.g., a glass orplastic bead, cap, or insert). Each body 300 then transfers the force F1exerted upon it by the linker 310 onto the front surface 117 of thereceptacle holder 110 opposite to the side surface 115 of the receptacleholder 110 that is in contact with the thermal element 200, therebyensuring maximal contact between the thermal element 200 and thereceptacle holder 110. In one embodiment, the apparatus 100 includes oneto ten linkers 310 per support 240, depending on the number ofreceptacle holders 110 positioned in sliding engagement therewith. Inanother embodiment, the apparatus 100 includes one linker 310 perreceptacle well 120. In yet another embodiment, the apparatus 100includes two linkers 310 per support 240. In yet another embodiment, theapparatus 100 includes five linkers 310 per support 240.

It is noted that the body 300 should have a lower thermal conductivitythan the receptacle holder 110 in order to prevent thermal energy fromtransferring from the receptacle holder 110 to the body 300 and/orsupport 240. Exemplary bodies for use in the apparatus include, but arenot limited to, glass or plastic beads that are connected, eitherdirectly or indirectly, to the receptacle holder by a linker 310 locatedbetween the support 240 and the body 300.

Thus, as shown in FIG. 7A, a support 240 of a row of receptacle holders110 may be provided as a solid surface by which body 300 and linker 310exert a force F1 to the receptacle holder 110 positioned in slidingengagement with the support 240 located in the row immediatelysubsequent thereto. For example, the body 300 of a first support 240positioned in a first row 101 of receptacle holders 110 exerts a forceF1 onto the front surface 117 of a receptacle holder 110 positioned insliding engagement with a second row 102, wherein the first 101 andsecond 102 rows are adjacent to one another within the apparatus 100, asdescribed herein.

In certain embodiments, the apparatus may include a single support 240in sliding engagement with all receptacle holders 110, or may include asingle support 240 in sliding engagement with each row (e.g., 101-106 inFIGS. 4 and 7A) of receptacle holders 110, or may include a singlesupport 240 in sliding engagement each individual receptacle holder 110.

Thermistors

In various embodiments, the apparatus may further include one or more(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) thermal sensors 610 tomonitor the temperature of the receptacle holder 110. A wide variety ofmicrosensors are available for determining temperatures, including,e.g., thermocouples having a bimetallic junction which produces atemperature dependent electromotive force (EMF), resistance thermometerswhich include material having an electrical resistance proportional tothe temperature of the material, thermistors, IC temperature sensors,quartz thermometers and the like. See, e.g., Horowitz and Hill, The Artof Electronics, Cambridge University Press 1994 (2nd Ed. 1994). As usedherein, the term “thermistor” refers to a type of resistor whoseresistance varies significantly with temperature. Such thermistors 610may be disposed in direct or indirect contact with the receptacle holder110. In one embodiment, two or more thermistors 610 are disposed incontact with a receptacle holder 110. In another embodiment, one or morethermistors 610 are disposed in contact with each of the receptaclewells 120 of a single receptacle holder 110 to enable monitoring of thetemperatures of each of the individual receptacle wells 120.

As shown in FIG. 5D, in an exemplary embodiment, receptacle holder 110may include a channel 612 disposed within the side surface 115. Invarious embodiments, the channel extends from an edge of the sidesurface 115 to a location corresponding a center-most receptacle well120 of the receptacle holder 110. For example, in embodiments whereinthe receptacle holder 110 includes five receptacle wells 120, thechannel 612 frequently extends to a location that corresponds with thethird/central receptacle well 120. The channel 612 is configured toaccept therein wires and/or electrical connections for the thermistors610 of the receptacle block. Disposing the wires and/or electricalconnections of the thermistors 610 within the channel 612 shields thethermistors and associated wiring thereof from the heat-depletingeffects of ambient temperatures, thereby ensuring accurate monitoring ofthe temperature of the receptacle holder 110. Provided at the end of thechannel 612 corresponding with the center-most receptacle well 120 maybe one or more closed through-holes 614 through which the thermistorsmay be provided such that the thermistors are in contact with thecenter-most receptacle well 120. In certain embodiments, twothrough-holes 614 are provided within the channel 612 such that twothermistors may be disposed therein to monitor the temperature of thereceptacle holder 110 on opposing sides of the center-most receptaclewells 120. Although not shown, channel 612 may extend the length of thereceptacle holder 110, and two closed through-holes 614 corresponding toeach of the receptacle wells 120 may be provided therein.

As shown in FIG. 5E, to accommodate the thermistors inserted throughthrough-holes 614, the receptacle holder 110 may be formed to includeone or more bulges 616 disposed on opposing sides of the center-mostreceptacle holder 120. The bulges 616 provide a closed-ended channelsurrounding a portion of the receptacle well 120 that extend from theside surface 115 of the receptacle holder 110 and terminating at thefront surface 117 opposite to the side surface 115 of the receptacleholder 110. Thus, when two thermistors 610 are provided to monitor thetemperature of the receptacle holder 110, one thermistor 610 is providedin each bulge 616 thereof. Although not shown, when channel 612 extendsthe length of the receptacle holder 110, and two through-holes 614 areprovided corresponding to each of the receptacle wells 120, receptacleholder 110 may include one bulge 616 per through-hole 614 to accommodateindividual thermistors therein. It is contemplated that additionalthrough-holes 614 may be provided, corresponding to two or more of thereceptacle wells, including one or more through-holes 614 for eachreceptacle well.

Heat Sink

In an exemplary embodiment, each support 240 is a heat sink or is inthermal communication with an individual heat sink 330. As used herein,the term “heat sink” refers to a component that transfers thermal energyfrom a higher temperature to a lower temperature fluid medium. The fluidmedium is frequently air but can also be water or in the case of heatexchangers, refrigerants and oil. A variety of suitable heat sinkconfigurations and related materials are well known in the art. As usedherein, the term “thermal communication” refers to the ability totransfer thermal energy from one body to another or from one body to afluid medium.

As shown in FIG. 7A, in certain embodiments, each support 240 isprovided in thermal communication with a single heat sink 330. Each heatsink 330 positioned in thermal communication with one or more supports240 of the apparatus 100, may further include a plurality ofthrough-holes 332 (see FIG. 7B) disposed in a surface thereof. Eachthough-hole 332 may be in direct alignment with the through-holes 242 ofthe support and/or the through-holes 170 at the bottom surface 160 ofthe receptacle holders 110 that are positioned in sliding engagementtherewith. Such through-holes 332 form a channel through which opticalfibers and/or associated components, for example, may pass therebyproviding optical communication between each receptacle well 120 and anexcitation signal source and/or an emission signal detector, asdiscussed below.

In certain embodiments, disposed within heat sink 330 may be one or morethermal devices separate from those used to heat the receptacle holdersto pre-heat the heat sink prior to the amplification assay. Pre-heatingof the heat sink may be desirable to reduce the temperature differencebetween the receptacle holder 110 undergoing heating and the heat sink,thereby avoiding sapping of the thermal energy being transferred to thereceptacle holder 110 by the thermal element 200. It has been found thatpre-heating the heat sink, among other things, improves temperaturecycling rates and reduces the electric and thermal strain on the thermalelement, thus reducing power consumption and increasing the lifespan ofthe thermal element. The heat sink may be pre-heated to a temperatureabove ambient temperature, but at or below a nucleic acid annealingtemperature, e.g., at or below about 50° C.-64° C., but above about 20°C.-22° C. In another embodiment, the heat sink may be pre-heated to atemperature between an annealing temperature and an elongation/extensiontemperature, e.g., between about 50° C.-64° C. and about 72° C.-80° C.In another embodiment, the heat sink may be pre-heated to a temperaturebetween an elongation/extension temperature and a melting/denaturationtemperature, e.g., between about 72° C.-80° C. to about 94° C.-98° C. Inanother embodiment, the heat sink may be pre-heated to a temperaturebetween an annealing temperature and a melting/denaturation temperature,e.g., between about 50° C.-64° C. and about 94° C.-98° C. Exemplarythermal devices used for pre-heating the heat sink 330 include, but arenot limited to, cartridge heaters 334. In various embodiments, the oneor more cartridge heaters 334 pre-heat the heat sink 330 to about 45°C.-50° C., for example, prior to the amplification assay. As should beunderstood, additional thermistors may be provided in thermal contactwith one or more portions of the heat sink to monitor the temperaturethereof to avoid sapping of the thermal energy being transferred to thereceptacle holder 110 by the thermal element 200.

Cover

As shown in FIGS. 1, 2, 8, and 12 , the apparatus 100 may also include acover 350 that is positioned in movable association with the receptacleholder 110. As can be expected, the cover 350 is movable between anopened position (FIG. 8B) and a closed position (FIG. 8C) relative tothe receptacle holder 110 and may be moved to any position betweenopened and closed, as necessary. In the opened position, the cover 350does not obstruct access to the receptacle wells 120 within thereceptacle holder 110 (see FIG. 8A). When in the closed position, thecover 350 will block and/or obstruct access to the receptacle wells 120.In addition, when closed, the cover 350 may exert a force F2 onto anyreceptacle within a receptacle well 120 to seat or secure the receptacle130 into the receptacle well 120 (see FIG. 8C). As discussed above,because the receptacle well 120 is configured to receive a receptacle130, the force F2 exerted by the cover 350 serves to ensure that thereceptacle 130 fits snugly within the receptacle well 120, therebyallowing maximal contact between the inner surface 180 of the receptaclewell 120 and the receptacle 130.

The cover 350 may be made from any rigid or semi-rigid material suitablefor exerting downward pressure onto a receptacle disposed within areceptacle well. Exemplary materials from which the cover may be madeinclude, but are not limited to, beryllium copper, spring steel, chromevanadium, chrome silicon, phosphor bronze, stainless steel, aluminum,titanium, tungsten, metal alloys, metal composites, plastic, or anysuitable rigid or semi-rigid material.

The cover 350 may be movable by any suitable mechanical element includedin the apparatus. In one embodiment, the cover 350 is hingedly attachedto the apparatus 100 so as to enable movement between the open andclosed positions. Attachment points include but are not limited to anyof the one or more supports of the apparatus or any suitable locationwithin a housing containing the apparatus. As shown in FIG. 1 , thecover 350 may be fixedly attached to a rigid rotatable member 352, whichis in movable communication with one or more electric motors 355. Therotatable member may be rotatably mounted to opposing sides of thehousing 50 of the apparatus or opposing sides of additional supportmembers thereof, and span a length of the apparatus parallel to theorientation of one or more receptacle holders such that actuation of therotatable member 352 results in the cover 350 being moved into theopened or closed position relative to the one or more receptacle holders110. In an exemplary embodiment, the rotatable member 352 is acylindrical rod having a circular cross-section and an axis of rotationat the center thereof, as shown in FIG. 8E, which is a sectional viewtaken along A′-A′ in FIG. 8D. Exemplary materials from which the rigidrotatable member may be made include, but are not limited to, steel,titanium, aluminum, or any suitable rigid material. As used herein, theterm “rotatably mounted” refers to any mounting orientation that allowsthe rotatable member to rotate about its center axis.

The cover 350 may comprise one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more) flexible extensions 360 attached to and extending laterallyaway from, the rigid rotatable member 352. Such flexible extensions 360are configured to make contact with at least a portion of a receptacle130 disposed within the receptacle holder 110 when the cover is in,approaching, or for a short distance after leaving, the closed position.As contact is made between the flexible extensions 360 and at least aportion of the receptacle 130, the flexible extensions 360 flex whileapplying force F2 directly to at least a portion of the receptacle 130.In an exemplary embodiment, the cover 350 includes two or more (i.e., 2,3, 4, 5, 6, 7, 8, 9, 10, or more) flexible extensions 360 extending inthe same direction away from the rigid rotatable member 352. In certainembodiments, the flexible extensions 360 extend laterally away from thehinged attachment of the cover 350 to the apparatus 100. In frequentembodiments, the cover 350 includes one flexible extension 360 perreceptacle well 120 of a receptacle holder 110. Also, in frequentembodiments, one flexible extension 360 of the cover 350 may contact atleast a portion of more than one receptacle 130 disposed within thereceptacle holder 110. Likewise, more than one flexible extension 360may contact at least a portion of more than one receptacle 130 disposedwithin the receptacle holder 110.

The cover 350 of the present disclosure often comprises multiplecomponents, such as flexible extensions 360, a rotatable member 352, orother elements, as a single molded cover unit, or in multiple elementscomprising the entire cover unit. For example, the flexible extensions360 may be attached to the rotatable member 352, or a single materialmay comprise the rotatable member 352 and the flexible extensions 360.

The apparatus 100 may include a single cover 350 in moveable associationwith all receptacle holders 110 (not shown), or it may include a singlecover 350 for each row of receptacle holders 110, or it may include asingle cover 350 for each individual respective receptacle holder 110.Movement of each cover 350 may be actuated by an electric motor 355disposed either within the apparatus 100 or within the housing 50 inwhich the apparatus is located. When the apparatus 100 includes morethan one cover 350, each cover 350 may be actuated by its own motor 355,or more than one cover 350 may be actuated by the same motor 355. Assuch, when the apparatus 100 includes more than one cover 350, eachcover 350 may move independent of the next and/or more than one cover350 may be moved simultaneously. One of skill in the art wouldappreciate that independent movement of multiple covers utilizing asingle motor may be provided through, for example, appropriate cammingof its connection to each cover. The electric motor 355 is electricallyconnected to a controllable power source 210 for applying a currentthereto. Control of the power source 210 can be carried out by anappropriately programmed controller 370 (such as a computer) which mayreceive signals from another processor 750 that controls the automatedprocess steps involved with temperature cycling processes.

Though several embodiments of the apparatuses and methods of the presentdisclosure include a cover, a cover is not required, and often notincluded or desired. For example, in particularly frequent embodiments,the receptacle holder does not have a cover in operable orientationtherewith. In such embodiments, receptacles are often held in place inthe receptacle holder, for example, by gravity, friction, and/or anothermode. When the apparatus is provided without a cover for thereceptacles, it will frequently comprise any of the configurations ofthe apparatus described herein, but lacking the cover, including allassociated instrumentation and mechanical and/or electrical elementsassociated therewith. Frequently, in such embodiments, a pipettor orreceptacle transport mechanism will have uninhibited access to thereceptacle holder to introduce or remove receptacles at will. An exampleof an apparatus without a cover is depicted in FIG. 14B. Such anapparatus could be readily attached to a heat sink, in communicationwith a detection system, and a power source and be fully operational. Infrequent embodiments multiple of these apparatuses are incorporated on asingle heat sink.

Optical Fibers

With reference now to FIGS. 9A-9C, the apparatus 100 further includes aplurality of (i.e., more than one) optical fibers 400 to provide opticalcommunication of a receptacle well with at least one of an excitationsignal source 500 and an emission signal detector 510 (see FIG. 10 ). Inone embodiment, the apparatus 100 includes one optical fiber 400 perreceptacle well 120. Thus, when the apparatus 100 includes tenreceptacles wells 120, at least ten optical fibers 400 will be providedto establish optical communication between the receptacle well 120 andone or more excitation signal sources 500 and/or one or more emissionsignal detectors 510.

As used herein, an “optical fiber” refers to a flexible, transparentfiber made of glass or plastic that functions as a waveguide to transmitlight between the two ends (i.e., the first end and the second end) ofthe fiber. Typically, optical fibers include a transparent coresurrounded by an opaque cladding material with a lower index ofrefraction and low to no autofluorescence characteristics. It should beunderstood that an optical pathway or assembly comprising the opticalfiber may optionally include one or more filters, lenses, aspheres,etc., to modify and/or focus and excitation or emission signals passingtherethrough. Optionally, the apparatus 100 may include an opticalinterface 440 between the first end 410 of each optical fiber 400 andthe receptacle 130 (see FIG. 9C). Such optical interface 440 may includea filter, lens, asphere, nose, cap, or any other element having desiredoptical properties. However, it should be understood that in variousembodiments, the interface 440 is not, and/or does not function as alens. Exemplary interfaces 440 useful in the apparatus include, but arenot limited to, glass or plastic balls, noses or caps covering the firstend 410 of the optical fiber 400, or any suitable optically clearmaterial.

The first end 410 of each of the plurality of optical fibers 400 isdisposed outside, within, or extending through a through-hole 170 of thereceptacle well 120, thereby providing optical communication with areceptacle well 120, and/or a receptacle 130 disposed within thereceptacle well 120. When disposed within the receptacle well 120, asshown in FIG. 9A, the first end 410 of the optical fiber 400 may bemoveable within the through-hole 170 of the receptacle well 120 relativeto the inner surface 180 thereof. A variety of means of movement of thefirst end 410 of the optical fiber 400 within the through-hole 170 arecontemplated. For example, the first end 410 of the optical fiber 400may extend into the receptacle well 120, and when a receptacle 130 isplaced within the well 120, the receptacle 130 contacts the first end410 of the optical fiber 400, thereby providing optical communicationbetween the receptacle 130 and the optical fiber 400. In an exemplaryembodiment, the presence of a receptacle 130 within the receptacle well120 will cause the optical fiber 400 to move within the through-hole 170(e.g., through the application of a direct force) in a directionopposite from the inner surface 180 of the receptacle well 120 such thatthe receptacle 130 can make maximal contact with the inner surface 180of the receptacle well 120 while maintaining optical communication withthe optical fiber 400, as shown in FIG. 9B. In another embodiment, thedownward force F2 exerted by the cover 350 and/or the flexibleextensions 360 of the cover 350 onto at least a portion of a receptacle130 disposed within a receptacle well 120 causes the optical fiber 400to move within the through-hole 170 when the receptacle 130 contacts theoptical fiber 400. In such embodiments, the receptacle 130 may apply aforce F3 to the first end 410 of optical fiber 400 in substantially thesame direction as the force F2 being applied to the receptacle, which isdisposed within the well such that the end 410 of the optical fiber 400moves within the well 120.

As is known in the art, optical fibers are rigid members, thereby havinga certain amount of inherent resilience to movement. Thus, one of skillin the art would understand that optical fibers useful in the apparatus100 should have sufficient rigidity to resist bending or otherwisedeforming within the receptacle holder 120 upon application of force F3onto the first end 410 thereof. Alternatively, a flexible optical fiber400 may be utilized, but the first end 410 of the optical fiber 410 maybe surrounded or protected by a rigid ferrule 450, for example, thatoptionally moves within the through-hole in response to the applicationor release of force F3.

Often, the first end 410 of each of the plurality of optical fibers 400,or an area 420 proximal to the first end 410 of each of the plurality ofoptical fibers 400, is connected, directly or indirectly, to arespective through-hole 170 of a receptacle well 120 with a resilientelement 600. The resilient element 600 thereby compresses and/or deformsas the optical fiber 400 moves within the through-hole 170 and returnsto its uncompressed and/or original form when the optical fiber 400returns to its rest position to thereby moderate movement of opticalfiber 400. As used herein, the “rest position” of an optical fiberrefers to the position of the first end 410 thereof when no receptacleis present within the receptacle well and/or when no downward force F2is exerted by the cover 350 onto at least a portion of a receptacle 130disposed within the receptacle well 120. Exemplary resilient elementsinclude, but are not limited to springs, plastics, opened- orclosed-cell foams, rubbers, dampers, pneumatic elements, hydraulicelements, electromagnetic elements, or combinations thereof.

One of skill in the art would understand that the inherentresilience/rigidity of the optical fiber 400 should be taken intoconsideration when selecting a resilient element 600 for use in theapparatus 100 to avoid having the optical fiber's inherent rigidityinterfere with the ability of its movement within the through-hole 170.Thus, in frequent embodiments, each optical fiber 400 in the apparatus100 has one or more dedicated resilient element(s) 600. Also, infrequent embodiments, two or more optical fibers 400 are in contact witha single resilient element 600 that permits individual or coordinatedmovement of the two or more optical fibers 400.

In yet another exemplary embodiment, the movement of the optical fiber400 within the through-hole 170 of the receptacle well 120 is associatedwith the movement of the cover 350 of the apparatus 100. For example, insuch embodiments the optical fiber 400 may be disposed outside, within,or extending through a through-hole 170 of the receptacle well 120 (asshown in FIG. 9A). Here the optical fiber 400 may, for example, be inmoveable connection with the motor 355 that actuates the cover 350 suchthat the same motor 355 actuates movement of the optical fiber 400within the through-hole 170. Alternatively, the optical fiber 400 may,for example, be in moveable connection with a motor (not shown) that isdifferent than the motor 355 that actuates the cover 350, but the actionof the motors on the cover 350 and optical fiber 400 may be coordinatedsuch that the optical fiber 400 moves within the through-hole 170 in atime period that corresponds to the movement of the cover 350. Thiscorresponding time period may comprise an overlapping time period ordistinct, but associated, time periods. For example, the fiber 400 maymove at the same time as the cover 350, the fiber 400 may move duringonly a portion of the time the cover 350 is moving, or the fiber 400 maymove during a time that is before or after movement of the cover 350. Infrequent, non-overlapping time period embodiments, before the cover 350begins to move toward the closed position, the first end 410 of theoptical fiber 400 may move within the through-hole 170 toward the innersurface 180 of the receptacle well 120. Alternatively, in otherfrequent, non-overlapping time period embodiments, before the first end410 of the optical fiber 400 begins to move toward the inner surface 180of the receptacle well 120, the cover 350 moves toward the closedposition. Often, however, the movement of the optical fiber 400 and thecover 350 is coordinated such that the first end 410 of the opticalfiber 400 moves toward the inner surface 180 of the receptacle well 120after the cover 350 has begun to move and is approaching a closedposition. In such embodiments, the first end 410 of the optical fiber400 can be actuated to move towards the interior of the receptacle well120 at the beginning of the movement of the cover 350 away from theclosed position, or at another time period.

In another exemplary embodiment, the rest position of the first end 410of the optical fiber 400 is below the inner surface 180 of thereceptacle well 120. In other words, the first end 410 of the opticalfiber 400 is at rest within the through-hole 170 of the receptacleholder 110. In such an embodiment, the first end 410 of the opticalfiber 400 is therefore moved towards the interior of the receptacle wellprior to, during, or after the cover 350 is moved to a closed positionin order to bring the first end 410 into contact with at least a portionof the receptacle 130 disposed within a receptacle well 120, orotherwise positioned close to, but not in direct contact with, a portionof the receptacle 130 in order to establish optical communicationtherewith. As discussed above, the optical fiber 400 may, for example,be in moveable connection with the motor 355 that actuates the cover 350such that the same motor 355 actuates movement of the optical fiber 400within the through-hole 170. Alternatively, the optical fiber 400 may,for example, be in moveable connection with a motor (not shown) that isdifferent than the motor 355 that actuates the cover 350, but the actionof the motors on the cover 350 and optical fiber 400 may be coordinatedsuch that the optical fiber 400 moves within the through-hole 170 in atime period that corresponds to the movement of the cover 350.

In yet another exemplary embodiment, movement of the optical fiber 400within through-hole 170 (either into or out of the interior ofreceptacle well 120) may be actuated through a mechanical connection tothe rigid rotatable member 352 of the cover 350. For example, a gearedor cammed mechanical connection (not shown) with the rigid rotatablemember 352 may be used to coordinate movement of the first end 410 ofthe optical fiber 400 into and away from the interior of the receptaclewell 120 as the cover 350 is moved into the opened or closed position.As such, the optical fiber 400 of the apparatus 100 may move into andout of the receptacle well 120 in conjunction with the opening andclosing of the cover 350 of the apparatus 100.

In certain embodiments, placement of a receptacle 130 within thereceptacle well 120 generally will not cause the optical fiber 400 tomove within the through-hole 170. However, as discussed above, the forceF2 exerted by the cover 350 onto at least a portion of the receptacle130 will prevent movement of the receptacle 130 within the receptaclewell 120, and allow for optical communication between the receptacle 130and the optical fiber 400, while maintaining maximal contact between thereceptacle 130 and the inner surface 180 of the receptacle well 120. Inembodiments wherein the rest position of the optical fiber 400 resultsin the first end 410 thereof being disposed below the inner surface 180of the receptacle well 120, force F2 maintains receptacle 130 in theseated position within receptacle well 120 even after the actuatedmovement of the optical fiber 400 into the interior of the receptaclewell 120. As should be understood, actuation of optical fiber 400 intocontact with receptacle 130 often exerts a force F4 (FIG. 9C) onto theclosed bottom end 138 of the receptacle 130. In the absence of force F2exerted by the cover 350 onto at least a portion of the receptacle 130,force F4 may dislodge or otherwise impair optimal contact of thereceptacle 130 with the inner surface 180 of the receptacle well 120.Therefore, in such embodiments, force F4 generally has the samemagnitude, or has a magnitude smaller than, force F2 or force F3.

Methods of Establishing Optical Communication

In another aspect, disclosed herein is provided a method forestablishing optical communication between a receptacle and anexcitation signal source and/or an emission signal detector within ahousing of the apparatus while allowing maximal contact between thesurface of the receptacle well and the receptacle (FIG. 11A). Asdiscussed in detail above, the method includes providing a receptacle130 to a receptacle well 120 of a receptacle holder 110 (step S110).Thereafter, a force F2 is applied to the receptacle 130 or at least aportion of the receptacle 130, such that the receptacle 130 fits snuglywithin the receptacle well 120, thereby allowing maximal contact betweenthe inner surface 180 of the receptacle well 120 and the receptacle 130(step S120). While force F2 is being applied to at least a portion ofthe receptacle 130, movement of a first end 410 of an optical fiber 400is effected toward the inner surface 180 of the receptacle well 120(step S130). In such embodiments, the receptacle 130 may apply a forceF3 to the optical fiber 400 in substantially the same direction as theforce F2 being applied to the receptacle 130, to the first end 410 ofthe optical fiber 400, which is disposed within the receptacle well 120such that optical communication is established between the bottom 138 ofthe receptacle 130 and the first end 410 of the optical fiber 400 (stepS140). As discussed above, movement of the first end 410 of the opticalfiber 400 is coordinated with movement of cover 350 into the closedposition. As such, the method may further include movement of the cover350 in coordination with the movement of the first end 410 of theoptical fiber 400.

Another exemplary embodiment of the method for establishing opticalcommunication between a receptacle and an excitation signal sourceand/or an emission signal detector within a housing of the apparatuswhile allowing maximal contact between the surface of the receptaclewell and the receptacle is shown in FIG. 11B. In this embodiment, themethod includes providing a receptacle 130 to a receptacle well 120 of areceptacle holder 110 (step S210). Thereafter, a cover 350 is moved intothe closed position (step S220), thereby exerting a force F2 onto thereceptacle 130 or at least a portion of the receptacle 130, such thatthe receptacle 130 fits snugly within the receptacle well 120, therebyallowing maximal contact between the inner surface 180 of the receptaclewell 120 and the receptacle 130 (step S230). Prior to, during, or aftermovement of the cover 350 into the closed position, movement of a firstend 410 of an optical fiber 400 is effected toward and into contact withthe seated receptacle 130 (step S240). Upon contact of the first end 410of the optical fiber 400 with the closed end 138 of the receptacle 130,force F4 is exerted by the first end 410 onto the receptacle 130. Insuch embodiments, the receptacle 130 may apply a force F3, which isgreater than force F4, onto the first end 410 of the optical fiber 400in substantially the same direction as the force F2. As such, opticalcommunication is established between the first end 410 of the opticalfiber and the receptacle 130, while ensuring maximal contact between thereceptacle 130 and the inner wall 180 of the receptacle well 120 (stepS250). As discussed above, movement of the first end 410 of the opticalfiber 400 is coordinated with movement of cover 350 into the closedposition.

Often, the first end 410 of each of the plurality of optical fibers 400,or an area 420 proximal to the first end 410 of each of the plurality ofoptical fibers 400, is connected, directly or indirectly, to arespective through-hole 170 of a receptacle well 120 with a resilientelement 600, as discussed above. The resilient element 600 therebycompresses and/or deforms as the optical fiber 400 moves within thethrough-hole 170 and returns to its uncompressed and/or original formwhen the optical fiber 400 returns to its rest position.

Stripper Plate

With reference now to FIGS. 12A-12D, the apparatus 100 may furtherinclude one or more stripper plates 650 mounted in moveable associationwith the receptacle holder 110. Like the cover 350, the stripper plate350 is movable between an opened and closed position, which in referenceto the stripper plate 650, will be referred to as the “unlockedposition” and the “locked position.” When in the unlocked position (FIG.12B), the stripper plate 650 permits transfer to and removal of areceptacle 130 into or out of a receptacle well 120. When in the lockedposition (FIG. 12C), the stripper plate 650 inhibits removal of areceptacle 130 disposed within a receptacle well 120, thereby permittinga receptacle transport mechanism 700, such as a pipettor orpick-and-place robot, to withdraw from receptacle 130 (FIG. 12D). Itshould be understood that when in the locked position, the stripperplate 650 does not inhibit access to the top of the receptacle 130 by areceptacle transport mechanism 700 used to deliver to and/or removereceptacles 130 but will prevent removal of the receptacle 130. Thus,removal of the receptacle 130 may only occur, if the stripper plate 650is present, when the stripper plate 650 is in the unlocked position.

When present, the stripper plate 650 may be made from any rigid materialsuitable for removing a receptacle 130 from a receptacle transportmechanism 700. Exemplary materials from which the stripper plate may bemade include, but are not limited to, beryllium copper, spring steel,aluminum, titanium, plastic, or any suitable rigid material.

In various embodiments, the apparatus 100 may include a single stripperplate 650 in moveable association with all receptacle holders 110, or itmay include a single stripper plate 650 for each row of receptacleholders 110, or it may include a single stripper plate 650 for eachindividual receptacle holder 110. Movement of the stripper plate 650 maybe actuated by an electric motor 660 disposed either within theapparatus 100 or within the housing 50 in which the apparatus islocated. When more than one stripper plate 650 is provided in theapparatus, each stripper plate 650 may be actuated by its own motor 660,or more than one stripper plate 650 may be actuated by the same motor660. As such, when the apparatus 100 includes more than one stripperplate 650, each stripper plate 650 may move independent of the nextand/or more than one stripper plate 650 may be moved simultaneously. Theelectric motors 660 effecting movement of the one or more stripperplates 650 are electrically connected to a controllable power source 210for applying a current thereto. Control of the power source 210 can becarried out by an appropriately programmed processor 670 (such as acomputer) which may receive signals from another processor that controlsthe automated process steps involved with thermal cycling processes.

As with movement of the optical fibers 400 discussed above, movement ofthe stripper plate 650 between the locked and unlocked positions may beassociated with the movement of the cover 350 of the apparatus 100. Forexample, in such embodiments the stripper plate 650 may be disposed inmoveable connection with the motor 355 that actuates the cover 350 suchthat the same motor 355 actuates movement of the stripper plate 650 asnecessary. Thus, the action of the motors on the cover 350 and stripperplate 650 may be coordinated such that the stripper plate 650 moves in atime period that corresponds to the movement of the cover 350. Thiscorresponding time period may comprise an overlapping time period ordistinct, but associated, time periods. For example, the stripper plate650 may move at the same time as the cover 350, the stripper plate 650may move during only a portion of the time the cover 350 is moving, orthe stripper plate 650 may move during a time that is before or aftermovement of the cover 350. However, movement of the stripper plate mustbe timed such that the receptacle transport mechanism 700 may withdrawfrom receptacle 130 without interfering with the movement of the cover350.

In other exemplary embodiments, the stripper plate 650 may be movablebetween the locked and unlocked positions by any suitable mechanicalelement included in the apparatus. In an exemplary embodiment, thestripper plate 650 is hingedly attached to the apparatus 100 so as toenable movement between the locked and unlocked positions. Attachmentpoints include, but are not limited to, any of the one or more supportsof the apparatus or any suitable location within a housing 50 containingthe apparatus 100. In another embodiment, the stripper plate 650 isslidingly attached to opposing sides of a support of the apparatus 100.For example, the stripper plate 650 may laterally slide in a directionperpendicular to the orientation of the rows (101-106) of receptacleholders 110. While a stripper plate may be utilized in certainembodiments described herein, it is often not incorporated as a featurewhen the receptacle transport mechanism 700 is provided with areceptacle release such as a tip stripper, ejection mechanism, or otherreceptacle release mechanism known in the art.

Second Exemplary Embodiment of the Apparatus

With reference now to FIGS. 14A-14D and 16 , there is provided a secondexemplary embodiment of the apparatus 800 described herein. Thedescription will be provided based on the differences from the firstexemplary embodiment discussed above. As such any reference to likeelements should be understood as described above.

As in the previous exemplary embodiment, the apparatus 800 includes oneor more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or any wholenumber between 1 and 20, or more) receptacle holders 110 (see also FIG.3 ). When multiple receptacle holders 110 are provided in an apparatusdescribed herein, each receptacle holder 110 disposed within theapparatus may be disposed in alignment with one another to facilitatethe automated processing steps involved in nucleic acid amplificationassays. Such an apparatus 800 may include a housing 50 (see FIGS. 2 and4 ) within which the one or more receptacle holders 110 are located. Thehousing 50 may be made from any suitable structural material such as,for example, plastic or metal.

As shown in FIG. 14A, the upright portion 247 of the support 240includes a mount 810 projecting from a third side thereof, forattachment to a controller board 820 shown in FIG. 14B. Attachment ofthe controller board 820 to the mount 810 of the support 240 may beaccomplished by any means known in the art. For example, the controllerboard 820 may be fixedly attached by rivets or screws 825. Often, theattachment is by way of a mechanism that permits independent lateralmovement between the support 240 and the controller board 820. Such anattachment may be by way of the use of shoulder screws or machining ofthe attachment points in the support 240 such that the controller boardcan move laterally over them regardless of the type of securing meansthat is utilized, including standard-threaded screws. Such lateralmovement of the controller board 820 facilitates the mounting of thesupport 240/controller board 820 unit to the heat sink 330, and allowsfor proper alignment of through-holes 242 of the support 240 withthrough-holes 332 of the heat sink 330, thereby providing optimalpositioning of optical fibers 400 for examination of the contents of areceptacle well 120.

The controller board 820 may include logic and control circuitry forperforming one or more of the motorization and temperature controlfunctions described above. In various embodiments, the controller board820 includes at least one electronic connection point 830 for electricalconnection to a second controller board 835 (see FIG. 16 ) disposed onthe apparatus 800. In frequent embodiments, the entire unit (orapparatus), including the circuitry on the controller board 820,depicted in FIG. 14B represents an independently calibrated unit thatcan be utilized when plugged into a power source and oriented incommunication with a detection system, such as an optical system oranother detection system. In such embodiments, the controller board 820has been configured to operate with the installed elements identified inFIGS. 14A-14D, with or without the primary or secondary cover 840/850,or any other cover means. In essence, the unit exemplified in FIG. 14Bmay be operated and utilized as a “plug-and-play” type of apparatus,whereby it can be installed, removed, or replaced with a different unit,at will, without the need for independent calibration of the overalldetection system after installation. As discussed above, disposedbetween the receptacle holder 110 and the upright portion 247 of thesupport 240 is a thermal element 200, such as a “Peltier device.” Acompressive housing 855 having a top surface 857 is configured forsecurable attachment over the receptacle holder 110. Disposed in the topsurface 857 of the compressive housing 855 are a plurality ofthrough-holes, each corresponding to and in alignment with a receptaclewell 120 of the receptacle holder 110. One or more cross-braces 248 aremounted to the support 240 and exert a force F1 (FIG. 5B) onto a sidesurface of the compressive housing 855, which in turn exerts the forceF1 onto the receptacle holder 110. The support 240 and compressivehousing 855 may each be formed from a material having low thermalconductivity such as plastic. In certain embodiments, the material fromwhich the support 240 and compressive housing 855 are formed may be thesame material or may be different materials.

FIG. 14D depicts a top view of a portion of the apparatus 800 providedin FIGS. 14A-14C. In various embodiments, the compressive housing 855has a side wall 864 with sloped ends 859 that are oriented toward thereceptacle holder 110. The sloped ends 859 enhance the uniformity of thecompressive force of the compressive housing 855 on the receptacleholder 110, while simplifying the compressive connection, installationease, and serviceability of the unit. In this embodiment, screws 827 maybe threaded through the upright portion 247 and into the cross-brace248. When the screws 827 are tightened, the cross-brace 248 exerts aforce on the outer surface of the side wall 864 (i.e., the surfacefacing away from the receptacle holder 110), and the outer ends 249 ofthe cross-brace 248 are pulled toward the upright portion 247, resultingin a bowed curvature (not shown) of the cross-brace 248 around thecompressive housing 855. The sloping ends 859 permit cross-brace 248bowing while enhancing the overall uniformity of the applied compressiveforce F1. The compressive housing 855 illustrated in FIG. 14D furtherincludes a plurality of ribs, including end ribs 861 and intermediateribs 862, extending from the inner surface of side wall 864 (i.e., thesurface facing the receptacle holder 110) and contacting the frontsurface 117 of the receptacle holder 110 adjacent the receptacle wells120. The end ribs 861 in the illustrated embodiment contact thereceptacle holder 110 at locations outside the endmost receptacle wells120, and the intermediate ribs contact the receptacle holder 110 betweenadjacent pairs of receptacle wells 120. One of skill in the art wouldappreciate that additional orientations and configurations of thecross-brace 248/compressive housing 855 connection could be providedwithout departing from the scope of the present disclosure.

With reference now to FIG. 16 , each support 240 may be a heat sink, maybe in thermal communication with an individual heat sink 330, or may bein thermal communication with a single heat sink 330. Each heat sink 330positioned in thermal communication with one or more supports 240 of theapparatus 800, may further include a plurality of through-holes 332 (seeFIG. 7B) disposed in a surface thereof. Each though-hole 332 may be indirect alignment with the through-holes 242 of the support and/or thethrough-holes 170 at the bottom surface 160 of the receptacle holders110 that are positioned in engagement therewith. Such through-holes 332form a channel through which optical fibers and/or associatedcomponents, for example, may pass thereby providing opticalcommunication between each receptacle well 120 and an excitation signalsource and/or an emission signal detector, as discussed above.

As shown in FIG. 14C, the apparatus 800 may also include a primary cover840 that is fixedly positioned over the receptacle holder 110. Theprimary cover 840 may be formed with one or more (i.e., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more) securing arms 845 in direct alignment with andcircumscribing each receptacle well 120 of the receptacle holder 110. Incertain embodiments, the primary cover 840 is formed with four securingarms 845 in direct alignment with and disposed in a surroundingarrangement with each receptacle well 120 of the receptacle holder 110.The securing arms 845 are configured for securable attachment to atleast a portion of the receptacle or cap 135 that is attached to areceptacle 130. Such securable attachment is analogous to the force F2exerted by the cover 350, as discussed above, for ensuring that thereceptacle 130 fits snugly within the receptacle well 120, therebyallowing maximal contact between the inner surface 180 of the receptaclewell 120 and the receptacle 130. The securing arms can be made of anysuitable material, including plastic, metal, or a metal composite.

The apparatus 800 may further include a secondary cover 850 fixedlypositioned over the primary cover 840. The secondary cover 850 may beformed with one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)releasing arms 856, each in direct alignment and in sliding contact withthe securing arms 845 of the primary cover. In various embodiments, thesecuring arms 845 of the primary cover include an angled surface 847upon which the corresponding releasing arm 856 of the secondary cover850 may slide when actuated during an automated process. The secondarycover 850 may further include one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) actuators 860 that are fixedly connected to thereleasing arms 856, and are positioned such that when a force is appliedthereon, the force is transferred from the actuator 860 to the releasingarms 856, which in turn, press onto the angled surface 847 of theprimary cover 840 and release the securable attachment to the cap 135that is attached to a receptacle 130.

It is therefore contemplated that the housing 50 within which theapparatus 800 is located will include at least one modified pipettor900, as shown in FIGS. 15A and 15B. As shown in FIG. 15A, the modifiedpipettor 900 is modified such that a plunger 910 is slidingly coupled toone or more limbs 915, which are hingedly attached to a body 920 of themodified pipettor 900. Thus, when the modified pipettor 900 causes theplunger 910 to be in a first position (as shown in FIG. 15A), the one ormore limbs 915 are in a retracted position such that a lower portion 917thereof are positioned in close proximity to the body 920. When themodified pipettor 900 causes the plunger 910 to be in a lowered secondposition (as shown in FIG. 15B), the one or more limbs 915 are thenmoved into an extended position such that the lower portions 917 thereofare moved away from the body 920.

This modified pipettor 900 is useful for engaging the secondary cover850 and pressing on the secondary cover 850 in a downward movement thatactuates the release of the securing arms 845 by the physical action ofthe releasing arms 856. When the releasing arms 856 are depressed inthis manner, the securing arms 845 are pulled axially away from thereceptacle 130 and cap 135, permitting its unencumbered release andlifting out by the pipettor. In such circumstances it is advantageousthat the releasing arms 856 maintain contact with, and depress in aradially outward manner, the securing arms 845 for the time periodrequired for the pipettor plunger 910 to frictionally engage thereceptacle cap and to lift the receptacle and cap vertically clear ofthe securing arms 845.

FIG. 16 depicts an alternate embodiment of a cover mechanism 824 that isactuated by an automated pipettor or a modified pipettor 900, asdescribed above. In this embodiment the end or plunger 910 of a pipettor900 contacts and depresses a cover release mechanism 852, which opensthe cover 822, permitting access of the receptacle 130 to the well 120.Once the receptacle 130 is placed in the well 120, the pipettor end orplunger is utilized to depress a cover securing mechanism 854, whichcauses a force to be exerted on the cap 135 and/or receptacle 130 whichthen is securely seated in the well 120. The force is analogous to theforce F2 exerted by the cover 350, as discussed above, for ensuring thatthe receptacle 130 fits snugly within the receptacle well 120, therebyallowing maximal contact between the inner surface 180 of the receptaclewell 120 and the receptacle 130. The cover 822 may be motor-actuated, asdiscussed above, may be actuated through one or more torsion springsdisposed on the rigid rotatable member 352 (FIG. 8B) to which the cover822 is fixedly attached, or may be actuated by a spring mechanism thatcauses the cover 822 to move vertically with respect to the receptacleholder 110. In various embodiments, the spring-loaded cover 822 mayinclude a push-lock fastener, which may lock the spring-loaded cover 822into the locked position. The push-lock mechanism may be directly orindirectly associated with the cover release mechanism 852 and/or thecover securing mechanism 854.

In certain embodiments, any of the apparatuses described herein will notinclude a cover or a mechanism to exert force F2 onto the cappedreceptacle 130. In such embodiments, the receptacle 130 fits snuglywithin the receptacle well 120, thereby allowing maximal contact betweenthe inner surface 180 of the receptacle well 120 and the receptacle 130without the need for a force F2.

In certain embodiments, any of the apparatuses described herein mayinclude a cover but will not include a mechanism to exert force F2 ontothe capped receptacle 130. In such embodiments, the cover does notcontact the capped receptacle, as the receptacle 130 fits snugly withinthe receptacle well 120, thereby allowing maximal contact between theinner surface 180 of the receptacle well 120 and the receptacle 130without the need for a force F2.

Use of the Apparatus in a Biochemical Assay

Use of the apparatus described herein is envisioned as, but is notlimited to, being part of an automated process for performing abiochemical assay, such as nucleic acid amplification. Thus, in anotheraspect, there is provided a method of conducting automated,random-access temperature cycling processes (see FIG. 13 ). A reactionmixture is prepared by first providing oil to a first receptacle 130 orfirst set of receptacles (step S310), reconstituting a PCR master mix(step S320) and providing the reconstituted PCR master mix to thereceptacle 130 (step S330). The sample to be assayed is thereafterinserted into the receptacle 130 containing the PCR master mix (stepS340), thereby forming a reaction mixture 140, and the receptacle 130 iscapped (step S350). The first receptacle 130 or first set ofreceptacles, each containing a reaction mixture 140 is transferred by areceptacle transport mechanism 700 to a first receptacle holder 110 ofthe apparatus 100 (step S360). The steps of preparing a reaction mixturemay optionally be repeated to fill a particular receptacle holder 110and/or a particular row (step S370).

If a cover 350 and/or stripper plate 650 associated with the particularreceptacle holder 110 is present and in the closed and/or lockedposition, the cover and/or stripper plate is moved to the opened and/orunlocked position to receive the first receptacle 130 or first set ofreceptacles. Alternatively, if the apparatus includes an alternativeembodiment of the cover 824, the receptacle transport mechanism 700, adidi (i.e., end of a pipettor), or a modified pipettor 900, may depressa cover releasing mechanism 852, thereby causing the cover 822 to bemoved to the opened position. Yet in another alternative embodiment, ifno cover is present, then any step involving movement of the cover isunnecessary and therefore may be omitted. Once the cover 350 and/orstripper plate 650 associated with the receptacle holder 110 is in theopened and/or unlocked position, the receptacle transport mechanism 700places the first receptacle 130 or first set of receptacles into one ormore receptacle wells 120 of the first receptacle holder 110 (stepS360). Prior to withdrawal of the receptacle transport mechanism 700, ifpresent, the stripper plate 650 is moved into the locked position toprevent removal of the transferred first receptacle 130, or set thereof,from the first receptacle holder 110 (not shown). In alternativeembodiments the receptacle transport mechanism 700 is provided with amechanism to remove receptacles without utilizing a stripper plate (see,e.g., U.S. Pub. No. 2010/0179687; U.S. Pub. No. 2005/0244303; U.S. Pat.Nos. 6,869,571; 6,824,024; and 6,431,015), thus rendering the use of astripper plate 650 or equivalent mechanism on the apparatus optional.The transferred receptacle 130 or set thereof may then be released fromthe receptacle transport mechanism 700 upon contact with the stripperplate 650 as the receptacle transport mechanism 700 withdraws therefrom.After the area surrounding the first receptacle holder is clear of thereceptacle transport mechanism, the cover 350 is moved into the closedposition (step S380). If the alternative embodiment of the covermechanism 824 is present in the apparatus, the receptacle transportmechanism 700, the didi, or the modified pipettor 900 depresses a coversecuring mechanism 854, thereby causing the cover 822 to be moved to theclosed position. As discussed above, once in the closed position, thecover may exert a force F2 onto at least a portion of the receptacle 130or a set of receptacles. However, in certain embodiments, the cover doesnot exert force F2 onto the receptacle 130.

As used herein, a “set” of receptacles refers to one or morereceptacle(s) 130 held within a receptacle holder 110. For example, a“set” of receptacles 130 refers to the number of receptacles 130required to at least partially, or to completely, fill a particularreceptacle holder 110. Thus, a set of receptacles 130 may refer to asingle receptacle 130 being processed by the apparatus 100, or it mayrefer to any whole number of receptacles 130 up to and including themaximum number of receptacle wells 120 within a particular receptacleholder 110.

The first receptacle holder 110 is then subjected to a first incubationprocess (step S390), which includes applying a voltage to a firstthermal element 200 of the apparatus 100 to alter the temperature of thefirst receptacle holder 110. By altering the temperature or temperaturesof the first receptacle holder 110, the first set of receptacles 130within the first receptacle holder 110, including the reactionmixture(s) 140 contained in each receptacle 130, is brought to apredetermined temperature and optionally sustained at the temperaturefor a predetermined time.

During the first incubation process, a second set of receptacles 130,each containing a reaction mixture 140, is optionally transferred by thereceptacle transport mechanism 700 to a second receptacle holder 110 ofthe apparatus 100 (step S400). As with the first set of receptacles, ifa cover 350 and/or stripper plate 650 associated with the secondreceptacle holder 110 is present and in the closed and/or lockedposition, the cover and/or stripper plate is moved to the openedposition and/or unlocked position to receive the second set ofreceptacles. Once the cover 350 and/or stripper plate 650 associatedwith the second receptacle holder 110 is in the open position and/orunlocked position, the receptacle transport mechanism 700 places thesecond set of receptacles into the receptacle wells 120 of the secondreceptacle holder 110. If a stripper plate 650 is utilized, prior towithdrawal of the receptacle transport mechanism 700, the stripper plate650 is moved into the locked position to prevent removal of thetransferred second set of receptacles from the second receptacle holder110. The transferred second set of receptacles 130 may then be releasedfrom the receptacle transport mechanism 700 upon contact with thestripper plate 650 as the receptacle transport mechanism 700 withdrawstherefrom. When the area surrounding the second receptacle holder 110 isclear of the receptacle transport mechanism 700, the cover 350associated therewith is moved into the closed position. As discussedabove, once in the closed position, the cover 350 exerts a force F2 ontoat least a portion of the respective set of receptacles.

The second receptacle holder 110 is then subject to a second incubationprocess, which may be the same as or different—in terms of temperatureand duration thereof—than the first incubation process. It should beunderstood that the first and second incubation processes may occursimultaneously or subsequent to one another. It should be furtherunderstood that a third or higher (i.e., third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, or higher) set ofreceptacles 130 may be transferred to the apparatus 100, which maythereafter, subject the third or higher set of receptacles 130 to athird or higher (i.e., third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, twelfth, or higher) incubation process. Suchadditional sets of receptacles 130 may be transferred and/or subject tothe additional incubation processes either simultaneously orsequentially, as necessary. The transfer of each subsequent set ofreceptacles 130 may be begun prior to completion of the incubationprocess for each immediately preceding set of receptacles 130.

In one exemplary embodiment, the first set of receptacles 130 is removed(step S410 of FIG. 13 ) from the first receptacle holder 110 immediatelyfollowing placement of the last of the second, or subsequent, set ofreceptacles 130 within the second receptacle holder 110. In a relatedexemplary embodiment, the second set of receptacles 130 is removed fromthe second receptacle holder 110 immediately following placement of thelast of the third, or subsequent, set of receptacles 130 within thesecond receptacle holder 110, and so forth. It will be appreciated bythose of skill in the art that the terms “first,” “second,” “third,” andhigher terms are relative terms, and are therefore not limited to thepositioning or orientation of the receptacle holders 110 within theapparatus 100. Similarly, it will be appreciated by those of skill inthe art that the terms “first,” “second,” “third,” and higher terms arenot limited to the timing of the incubation process relative to thetiming that the apparatus 100 is set up. These terms are merely intendedto be relative to the timing of placement and incubation of anyparticular set of receptacles 130 within each respective receptacleholder 110. For example, the 50^(th) set of receptacles may beconsidered to be the first set of receptacles relative to the 51^(st)set of receptacles.

Use of the second exemplary embodiment of the apparatus 800 describedherein, like the first exemplary apparatus, is also envisioned as, butis not limited to, being part of an automated process for performing abiochemical assay, such as nucleic acid amplification. As above, if nocover, primary cover, or secondary cover is present, then any stepinvolving movement of the covers is unnecessary and therefore may beomitted. As above, a reaction mixture is prepared by first providing oilto a first receptacle 130 or first set of receptacles (step S310),reconstituting a PCR master mix (step S320) and providing thereconstituted PCR master mix to the receptacle 130 (step S330). Thesample to be assayed is thereafter inserted into the receptacle 130containing the PCR master mix (step S340), thereby forming a reactionmixture 140, and the receptacle 130 is capped (step S350). The firstreceptacle 130 or first set of receptacles, each containing a reactionmixture 140 is transferred by a receptacle transport mechanism 700 to afirst receptacle holder 110 of the apparatus 100 (step S360). The stepsof preparing a reaction mixture may optionally be repeated to fill aparticular receptacle holder 110 and/or a particular row (step S370).Transferring of the first receptacle 130 or first set of receptacles isaccomplished by first performing an automated motion of moving theplunger 910 of a modified pipettor 900 into the raised position (asshown in FIG. 15A), thereby causing the knob 912 to slidingly contactthe one or more limbs 915 and move the limbs 915 into a retractedposition such that a lower portions 917 thereof are placed in closeproximity to the body 920 of the pipettor 900. The pipettor 900, havinga capped first receptacle 130 or first set of receptacles frictionallyattached thereto, places the first receptacle 130 or first set ofreceptacles into one or more receptacle wells 120 of the firstreceptacle holder 110 (step S360). As the first receptacle 130 or firstset of receptacles is lowered into the receptacle well 120, the firstreceptacle 130 or first set of receptacles contacts at least a portionof the securing arms 845 of the primary cover 840. The downward forceapplied by receptacle to the securing arms 845 cause the securing arms845 to flex in a radial outward direction in relation to the axialcenter of the receptacle well 120 such that the lower portion of the capclears the securing arms 845. Once the lower portion of the cap 135clears the securing arms 845, the securing arms return to their restposition, thereby securably contacting at least a portion of the cap135, ensuring that the capped receptacle fits snugly within thereceptacle well 120, thereby allowing maximal contact between the innersurface 180 of the receptacle well 120 and the receptacle 130.

The first receptacle holder 110 is then subjected to a first incubationprocess (step S390), which includes applying a voltage to a firstthermal element 200 of the apparatus 100 to alter the temperature of thefirst receptacle holder 110. By altering the temperature or temperaturesof the first receptacle holder 110, the first set of receptacles 130within the first receptacle holder 110, including the reactionmixture(s) 140 contained in each receptacle 130, is brought to apredetermined temperature and optionally sustained at the temperaturefor a predetermined time or fluctuated between a series of temperatures.

As above, during the first incubation process, a second set ofreceptacles 130, each containing a reaction mixture 140, is optionallytransferred by the receptacle transport mechanism 700 to a secondreceptacle holder 110 of the apparatus 100 (step S400).

Upon completion of the incubation process, removal of the firstreceptacle 130 or set of receptacles from the second exemplaryembodiment of the apparatus 800 is accomplished by performing anautomated motion of moving the plunger 910 of a modified pipettor 900into the lowered position (as shown in FIG. 15B), thereby causing theknob 912 to slidingly contact the one or more limbs 915 and move thelimbs 915 into an extended position such that a lower portions 917thereof are moved away from the body 920. Thereafter, the modifiedpipettor 900 is lowered into the open end of the cap 135 of the firstreceptacle 130 or set of receptacles. As shown in FIG. 15C, uponlowering of the modified pipettor 900, the extended lower portions 917of the limbs 915 contact the actuators 860 of the secondary cover 850and apply a downward force thereto. The downward force causes thereleasing arms 856 to slidingly contact the securing arms 845 of theprimary cover 840, causing the securing arms 845 to flex in a radialoutward direction relative the axial center of the receptacle well 120,as the modified pipettor 900 frictionally engages the open end of thecap 135. The flexing of the securing arms 845 releases the cappedreceptacle 130/135 or set of receptacles, allowing the capped receptacleto be removed from the receptacle well 120 as the modified pipettor 900is raised therefrom.

In certain embodiments, it is desirable to preheat the heat sink 330 ofthe apparatus 100 prior to or during the incubation process. In thoseembodiments, prior to, during, or after the first receptacle 130 orfirst set of receptacles, each containing a reaction mixture 140 istransferred by a receptacle transport mechanism 700 to a firstreceptacle holder 110 of the apparatus 100, a voltage is applied to athermal element 334 that is in thermal communication with the heat sink330. As discussed above, the heat sink may be warmed to about 45-50° C.,for example, prior to the biochemical assay. The transferred receptacle130 or set thereof may then be released from the receptacle transportmechanism 700 upon contact with the stripper plate 650 as the receptacletransport mechanism 700 withdraws therefrom. After the area surroundingthe first receptacle holder is clear of the receptacle transportmechanism, the cover 350 is moved into the closed position. As discussedabove, once in the closed position, the cover exerts a force F2 onto atleast a portion of the receptacle 130 or a set of receptacles.Thereafter, the first receptacle holder 110 is then subjected to a firstincubation process, which includes applying a voltage to a first thermalelement 200 of the apparatus 100 to alter the temperature of the firstreceptacle holder 110. By altering the temperature or temperatures ofthe first receptacle holder 110, the first set of receptacles 130 withinthe first receptacle holder 110, including the reaction mixture(s) 140contained in each receptacle 130, is brought to a predeterminedtemperature and optionally sustained at the temperature for apredetermined time.

In various embodiments, the temperature of the receptacle holder 110will be above ambient temperature as a result of a prior incubationprocess performed on a previous receptacle 130 or set thereof, or due topre-heating of the heat sink 330. In these embodiments, pre-heating oradditional heating of the heat sink 330 may or may not be desired due toa lessened risk of heat sap, as discussed above.

Each of the first and second sets of receptacles 130 (and/or anyadditional sets of receptacles) may be transferred to the apparatus 100by a single receptacle transport mechanism 700 or may be transferred bymore than one receptacle transport mechanism 700, depending on theconfiguration of the apparatus 100, system, or biochemical instrument.

Each set of receptacles 130 may undergo a single incubation process or aplurality of incubation processes prior to completion of the biochemicalassay. Alternatively, or in conjunction, each set of receptacles 130 mayundergo a single temperature ramp for purposes of, for example, a meltcurve analysis. If a set of receptacles 130 undergoes multipletemperature cycles, each subsequent temperature cycle may be the same asor different from the temperature cycle immediately preceding it. Duringthe single or plurality of incubation processes, during the temperatureramp, or upon completion of a predetermined number of incubationprocesses, an excitation signal source 500 transmits an excitationsignal via optical fibers 400 of the apparatus 100 to the set ofreceptacles 130. Any emission signals resulting therefrom are thereaftertransmitted via the optical fibers 400 to one or more emission signaldetectors 510. Though FIG. 10 depicts separate optical fibers branchingout to the excitation signal source 500 and emission signal detector510, various embodiments of the present disclosure utilize a singleoptical fiber (i.e., a light pipe) between the excitation signal source500 and its corresponding emission signal detector 510. One of skill inthe art will appreciate that a collection of mirrors, dichroics, and/orfilters can be utilized to split the excitation and emission signalstravelling through the single optical fiber between the excitationsignal source 500 and its corresponding emission signal detector 510. Inthese embodiments, one end of the single optical fiber terminates in orat a single receptacle well, and the other end of the single opticalfiber terminates at location in optical communication with theexcitation signal source 500 and its corresponding emission signaldetector 510. Often, in such a configuration, every receptacle well inthe apparatus will be outfitted with a similar optical fiberarrangement.

Following completion of all incubation processes and/or detection steps,the respective set of receptacles 130 is removed from the respectivereceptacle holder 110 of the apparatus 100. Removal of a set ofreceptacles 130 often proceeds as follows. The cover 350, if present,that is associated with the receptacle holder 110 in which the assayedreceptacles 130 are seated is moved to the opened position. Eithersimultaneously or shortly thereafter, the stripper plate 650, if presentand in the locked position, is moved to the unlocked position. Thereceptacle transport mechanism 700 is moved into position and loweredtoward the receptacle holder 110 to contact the tops of each of thereceptacles 130 seated therein. In frequent embodiments, the receptacletransport mechanism 700 contacts a single receptacle 130 at anyparticular time. In certain embodiments the receptacle transportmechanism 700 is capable of contacting and removing a set of receptacles130. Upon withdrawal of the receptacle transport mechanism 700, anyreceptacles contacted therewith are removed from the receptacle holder110. It should be understood that assayed receptacles 130 or setsthereof, may be removed prior to, during, or after completion of thetemperature cycling process of any previous or subsequent sets ofreceptacles.

Thus, a first set of receptacles may be removed from the firstreceptacle holder 110 prior to completion of the second incubationprocess of the second set of receptacles 130 within the secondreceptacle holder 110. Likewise, a second set of receptacles 130 may beremoved from the second receptacle holder 110 prior to completion of athird or higher incubation process of a third or higher set ofreceptacles 130 within the third or higher receptacle holder 110.

Because the present apparatus 100, 800 is capable of simultaneouslyconducting a variety of different assays, it is also envisioned that dueto the requirements of a particular assay, sample, reagents, or anyother reason, a second set of receptacles 130 may be subjected to ashorter incubation process than a first set of receptacles 130 such thatthe second or subsequent set of receptacles may be removed prior toremoval of the first set of receptacles.

Accordingly, the apparatus 100, 800 described herein provides theability to automate incubation processes, simultaneously involving thesame or different biochemical assays. In an exemplary embodiment, theapparatus includes six rows (101-106) of receptacle holders 110 with tworeceptacle holders 110 per row and five receptacle wells 120 perreceptacle holder 110. As such, the apparatus of the exemplaryembodiment is capable of simultaneously incubating up to sixtyreceptacles 130 at any given time. Assuming an incubation time of sixtyminutes for each set of receptacles 130 within each receptacle holder,and population of each receptacle holder 110 (containing five receptaclewells 120) with a set of receptacles 130 every five minutes, the firstset of receptacles will complete the incubation, about sixty fiveminutes after the first receptacle 130 is placed in a receptacle well120. Thereafter, every five minutes another set of five receptacles willcomplete its incubation period. When each set of receptacles hascompleted its incubation, the set is removed from the receptacle holderand replaced with a fresh set of receptacles for another incubationperiod. Thus, when an apparatus 100, 800 is used in conjunction with anautomated instrument for performing a biochemical assay, such as PCR,the apparatus increases instrument throughput productivity within atypical 8-hour shift.

System for Automated Random-Access Incubation

In another aspect, the present disclosure provides a system forautomated random-access incubation for nucleic acid amplificationassays. The system includes one or more of the apparatus 100, 800 andallows for simultaneous or individualized assays to be performed. Thesystem includes a housing 50 into which the one or more apparatuses 100,800 are located. As discussed above, the thermal element 200corresponding to each receptacle holder 110 may be independentlycontrollable to only alter the temperature of its correspondingreceptacle holder 110. Thus, the system may include more than onecontroller 220, each of which is electrically connected to a singlethermal element 200 and one or more thermistors 610 of an individualreceptacle holder 110, and/or connected to a controllable power source212 connected to a motor 355 effecting movement to a cover 350, ifpresent, and/or a motor 660 effecting movement of a stripper plate 650,if present, of the individual receptacle holder 110. It should beunderstood that any one or more controllers (220, 370, 670) may becombined to effect independent control of more than one thermal element200 and/or controllable power source 212 connected to an electric motor(355, 660). Thus, the system may include a single controllerelectrically connected to each of the thermal elements 200 and to one ormore motors 355 disposed in moveable communication with the cover 350and/or stripper plate 650 corresponding to each receptacle holder 110.Likewise, the system may include an appropriately programmed processor750 (such as a computer) which is electrically connected to eachcontroller (220, 370, 670) to send and/or receive signals/commands forperforming an incubation process. In certain embodiments, the controller(220, 370, 670) and the processor 750 will be configured in the sameunit, thereby reducing the number of components within the system.

As discussed above, the system will include at least one heat sink 330.Thus, each of the one or more apparatuses 100, 800 within the system maybe disposed in independent thermal communication with a single heat sink330 (i.e., one heat sink per apparatus), or every apparatus 100, 800 maybe in thermal communication with a single heat sink 330. In certainembodiments, each receptacle holder 110 of each apparatus 100, 800 ofthe system will be disposed in independent thermal communication with adedicated heat sink 330 (i.e., one heat sink per receptacle holder), asdiscussed above.

As shown in FIG. 10 , the system may further include one or moreexcitation signal sources 500 and one or more emission signal detectors510 to which the second ends 430 of the optical fibers 400 of theapparatus 100, 800 included therein are in optical communication.Excitation signal sources 500 and emission signal detectors 510contemplated by the present disclosure include, but are not limited to,fluorometers, luminometers, spectrophotometers, infrared detectors andcharged-coupled devices. Each of these types of optical detectionsystems can be positioned within the housing of the apparatus 100, 800,within the housing of the system, or within the overall housing of thebiochemical analysis instrument, as appropriate. Multiple types ofsignal sources 500 and signal detectors 510 may be movably mounted on aplatform to facilitate different detection methods for differentprocesses. The system may also include multiple detectors of the same ordifferent types for detecting signals emitted from different receptacles130 simultaneously. As discussed above, though FIG. 10 depicts separateoptical fibers branching out to the excitation signal source 500 andemission signal detector 510, certain embodiments of the presentdisclosure utilize a single optical fiber (e.g., a light pipe) betweenthe excitation signal source 500 and its corresponding emission signaldetector 510.

The system may further include a receptacle transport mechanism 700(e.g., a pick-and-place mechanism, a pipettor, or a modified pipettor)that is positioned within the housing of the system or within theoverall housing of the instrument. The receptacle transport mechanism700 is configured to transfer and/or remove one or more receptacles 130,either individually or as a set, from a receptacle holder 110 of theapparatus 100. In various embodiments, the modified pipettor 900includes a body having a plunger slidingly disposed therein, and one ormore limbs hingedly attached to the body and positioned in slidingcommunication with a knob fixedly attached to the plunger. When theplunger is in a first position, a lower portion of the one or more limbsare proximal to the body, and when the plunger is in a second position,the lower portion of the one or more limbs are extended in a radialoutward direction relative to the body. In certain embodiments, thereceptacle transport mechanism 700 is configured to additionallydispense into and/or remove liquids from individual receptacles 130.

Although the present disclosure has been described with reference to theabove example, it will be understood that modifications and variationsare encompassed within the spirit and scope of the disclosed subjectmatter. Accordingly, the present disclosure is limited only by thefollowing claims.

What is claimed is:
 1. An apparatus for performing nucleic acidamplification reactions, comprising: a thermally-conductive receptacleholder having a front surface and a back surface, the front surfacecomprising a row of receptacle wells, wherein an outer surface of eachof the receptacle wells has a frustoconical shape, wherein an innersurface of each of the receptacle wells is defined to receive areceptacle, and wherein each of the receptacle wells has a through-holeextending between the inner and outer surfaces thereof; one or morethermal elements for altering a temperature(s) of the receptacle holder,the one or more thermal elements being in contact with the back surfaceof the receptacle holder; and a plurality of optical fibers, each of theoptical fibers providing optical communication between one of thereceptacle wells and at least one of an excitation signal source and anemission signal detector, wherein a first end of each of the opticalfibers is disposed outside, within, or extends through the through-holeof a corresponding one of the receptacle wells, and wherein a second endof each optical fiber is in optical communication with the excitationsignal source and/or the emission signal detector.
 2. The apparatus ofclaim 1, further comprising two or more thermistors disposed in contactwith the receptacle holder.
 3. The apparatus of claim 2, wherein the twoor more thermistors comprise one or more thermistors disposed in contactwith each of the receptacle wells.
 4. The apparatus of claim 2, furthercomprising a channel disposed within the back surface and containingwires and/or electrical connections for the two or more thermistors. 5.The apparatus of claim 4, wherein the channel extends from an edge ofthe back surface to a location corresponding to a center-most receptaclewell of the receptacle holder.
 6. The apparatus of claim 5, furthercomprising a pair of closed through-holes provided at the location ofthe channel corresponding to the center-most receptacle at an end of thechannel, the closed through-holes being disposed on opposite sides ofthe center-most receptacle, wherein one of the two or more thermistorsis provided through each of the closed through-holes.
 7. The apparatusof claim 6, further comprising a pair of bulges disposed on oppositesides of the center-most receptacle well, wherein each of the bulges isconfigured to accommodate one of the two or more thermistors insertedthrough the closed through-holes.
 8. The apparatus of claim 5, whereinthe two or more thermistors consist of two thermistors.
 9. The apparatusof claim 1, further comprising a support, the support being coupled to alateral side surface of each of the one or more thermal elements, suchthat the one or more thermal elements are positioned between a portionof the support and the back surface of the receptacle holder.
 10. Theapparatus of claim 9, wherein the support comprises: a base portionhaving a plurality of through-holes, each of the through-holes beingaligned with the through-hole of one of the receptacle wells; and anupright portion extending from the base portion and coupled to thelateral side surface of the thermal element.
 11. The apparatus of claim10, further comprising a heat sink coupled to the base portion of thesupport, wherein the heat sink comprises a plurality of through-holesaligned with the plurality of through-holes of the base portion of thesupport.
 12. The apparatus of claim 1, further comprising a controllerelectrically connected to the one or more thermal elements to cycle thetemperature of each of the one or more thermal elements.
 13. Theapparatus of claim 1, wherein the first end of each optical fiber isfixedly disposed within the through-hole of the corresponding receptaclewell.
 14. The apparatus of claim 1, further comprising a cover movablebetween an open position and a closed position relative to thereceptacle holder, wherein one or more receptacles disposed within oneor more of the receptacle wells are seated or secured within thereceptacle well by the cover when the cover is moved from the openposition to the closed position, and wherein the first end of eachoptical fiber moves within its corresponding through-hole when (1) thecover is moved to the open or closed position, or (2) a receptacle ispresent in the receptacle well and the cover is moved to the open orclosed position.
 15. The apparatus of claim 1, wherein the apparatusdoes not include a cover.